In situ-generated microfluidic isolation structures, kits and methods of use thereof

ABSTRACT

In situ-generated microfluidic isolation structures incorporating a solidified polymer network, methods of preparation and use, compositions and kits therefor are described. The ability to introduce in real time, a variety of isolating structures including pens and barriers offers improved methods of micro-object manipulation in microfluidic devices. The in situ-generated isolation structures may be permanently or temporarily installed.

This application is a non-provisional application claiming the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application No. 62/258,957filed on Nov. 23, 2015, and of U.S. Provisional Application No.62/423,627 filed on Nov. 17, 2016, each of which disclosures is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

In biosciences and related fields, it can be useful to have the abilityto re-configure the flow region of a microfluidic device in real time.Some embodiments of the present invention include apparatuses andprocesses for in situ-generation of microfluidic isolation structures.

SUMMARY OF THE INVENTION

In one aspect, a microfluidic device is provided which includes anenclosure having: a substrate; a flow region located within theenclosure; and at least one in situ-generated isolation structuredisposed on the substrate, where the at least one in situ-generatedisolation structure includes a solidified polymer network. In someembodiments, the solidified polymer network does not include a siliconepolymer. In some embodiments, the solidified polymer network does notinclude silicon. The solidified polymer network may include aphotoinitiated polymer. All or at least part of the at least one insitu-generated isolation structure may consist of the solidified polymernetwork.

In another aspect, a microfluidic device is provided which includes anenclosure having: a substrate; a microfluidic channel; at least onesequestration pen; and an in situ-generated barrier. The sequestrationpen may include an isolation region and a connection region, theconnection region having a proximal opening to the microfluidic channeland a distal opening to the isolation region. The in situ-generatedbarrier may be disposed to provide at least a partial blockade of themicrofluidic channel and/or the sequestration pen. In variousembodiments, the in situ-generated barrier may include a solidifiedpolymer network. The solidified polymer network may include aphotoinitiated polymer.

In yet another aspect, a microfluidic device is provided which includesan enclosure having: a substrate; a flow region including a microfluidicchannel configured to contain a fluidic medium; a first plurality ofsequestration pens disposed adjacent to each other such that eachsequestration pen of the first plurality opens off a first side of themicrofluidic channel; and a second plurality of sequestration pensdisposed adjacent to each other such that each sequestration pen of thesecond plurality opens off a second opposing side of the microfluidicchannel. The first side of the microfluidic channel may be configured toreceive a first fluidic medium, and the second side of the microfluidicchannel may be configured to receive a second fluidic medium. In variousembodiments, the enclosure may further comprise a barrier. The barriermay be configured to divide the microfluidic channel into a firstsub-channel and a second sub-channel, wherein the first sub-channel isconfigured to provide a first sub-flow of fluidic medium past the firstplurality of sequestration pens, and wherein the second sub-channel isconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of sequestration pens. The barrier may comprise,consist of, or consist essentially of an in situ-generated barrier.Thus, in certain embodiments, the barrier may comprise a portion that isnot an in situ-generated barrier. In various embodiments, the barriermay be punctuated by at least one gap. The gap may be aligned between aproximal opening of a first pen of the first plurality of pens and aproximal opening of a corresponding first pen of the second plurality ofpens. In various embodiments, the in situ-generated barrier may preventcells from moving from the first sub-channel to the second sub-channel,and vice versa.

In another aspect, a method of isolating a micro-object in amicrofluidic device is provided, including the steps of: introducing afirst fluidic medium including a plurality of micro-objects into anenclosure of the microfluidic device, the enclosure including asubstrate and a flow region; introducing a solution including a flowablepolymer into the enclosure; activating solidification of the flowablepolymer at at least one selected area of the flow region, therebyforming an in situ-generated isolation structure; and isolating at leastone of the plurality of micro-objects with the in situ-generatedisolation structure. In some embodiments, the step of initiatingsolidification of the flowable polymer may include opticallyilluminating the at least one selected area of the flow region. In someembodiments, the solidification of the flowable polymer may include thepolymerization of polymers of the flowable polymer into a polymernetwork. The step of introducing a flowable polymer may further includeintroducing a photoactivatable polymerization initiator.

In another aspect, a method of isolating a micro-object in amicrofluidic device is provided, including the steps of: providing amicrofluidic device including an enclosure having a substrate, a flowregion including a microfluidic channel, and a plurality ofsequestration pens; introducing a first fluidic medium including aplurality of micro-objects into the microfluidic channel of themicrofluidic device; disposing ones of the plurality of micro-objects inat least a portion of the plurality of sequestration pens, therebyforming a plurality of populated sequestration pens each containing atleast one micro-object; introducing a second fluidic medium into themicrofluidic channel, wherein the second fluidic medium comprises aflowable polymer; selecting at least one of the plurality of populatedsequestration pens; initiating polymerization of the flowable polymer atat least a first selected point within or adjacent to at least oneselected sequestration pen, where the polymerized polymer of theflowable polymer generates at least a partial in situ-generated barrierthat prevents the at least one micro-object from exiting the at leastone selected sequestration pen. Each of the plurality of sequestrationpens may include an isolation region and a connection region, theconnection region having a proximal opening to the microfluidic channeland a distal opening to the isolation region. In certain embodiments,initiating polymerization of the flowable polymer at a selected pointwithin or adjacent to a sequestration pen comprises: initiatingpolymerization within the connection region or isolation region of thesequestration pen; and/or initiating polymerization at or adjacent to anedge of the proximal opening of the connection region.

In another aspect, a method of concentrating micro-objects in amicrofluidic device may be provided, including the steps of: providing amicrofluidic device including an enclosure having a substrate and a flowregion configured to contain a fluidic medium; introducing an insitu-generated isolation structure in a first sector of the flow region,where the in situ-generated isolation structure is configured to permitthe fluidic medium to flow through the in situ-generated isolationstructure while preventing at least one micro-object in the fluidicmedium from passing into, out of, and/or through the in situ-generatedisolation structure; introducing a first plurality of micro-objects in afirst volume of the fluidic medium into the first sector of the flowregion; and concentrating at least a first subset of the first pluralityof micro-objects in the first sector of the flow region. In variousembodiments, the first volume of the fluidic medium may be larger than avolume of the first sector of the flow region.

In another aspect, a method of assaying a cell of a clonal population ina microfluidic device is provided, the method including the steps of:introducing a first fluidic medium comprising a plurality of cells intoan enclosure of the microfluidic device, the enclosure comprising asubstrate, a flow region comprising a microfluidic channel configured tocontain a fluidic medium, a first plurality of sequestration pensdisposed adjacent to each other such that each sequestration pen of thefirst plurality opens off a first side of the microfluidic channel, anda second plurality of sequestration pens disposed adjacent to each othersuch that each sequestration pen of the second plurality opens off asecond opposing side of the microfluidic channel; flowing the firstfluidic medium and the plurality of cells into the microfluidic channelof the microfluidic device; introducing a clonal population of cells ineach of the sequestration pens of the first plurality of sequestrationpens; for each clonal population of cells in the first plurality ofsequestration pens, moving at least one cell to a respectivesequestration pen of the second plurality of sequestration pens;introducing a flowable polymer into the microfluidic channel; activatingsolidification of the flowable polymer along a length of themicrofluidic channel, thereby forming an in situ-generated barrierdividing the microfluidic channel into a first sub-channel configured toprovide a first sub-flow of fluidic medium past the first plurality ofsequestration pens and a second sub-channel configured to provide asecond sub-flow of fluidic medium past the second plurality ofsequestration pens, wherein the in situ-generated barrier prevents cellsfrom moving from the first sub-channel to the second sub-channel, andvice versa; flowing a second fluidic medium into the second sub-channel,wherein the second fluidic medium comprises reagents for assaying thecells in the second plurality of sequestration pens; and assaying thecell(s) in each sequestration pen of the second plurality. The step ofintroducing the clonal population may include introducing a single cellinto each of the first plurality of sequestration pens, and may furtherinclude expanding the single cell to a clonal population of cells.

In another aspect, a kit for isolating a micro-object within amicrofluidic device is provided, the kit including a microfluidic devicecomprising an enclosure having: a substrate and a flow region locatedwithin the enclosure; and a flowable polymer solution, where the polymermay be capable of polymerization and/or thermally-induced gelling. Insome embodiments, flowable polymer may be configured to be polymerizedby photoinitiation. In some embodiments, the kit may further include aphotoactivatable polymerization initiator. In some embodiments, themicrofluidic device may further include at least one sequestration pen.In various embodiments, the at least one sequestration pen may includean isolation region and a connection region, the connection regionhaving a proximal opening to the flow region and a distal opening to theisolation region.

In another aspect, a kit for assaying cells of a clonal population isprovided, the kit including: a microfluidic device comprising anenclosure having a substrate, a flow region including a channel locatedwithin the enclosure, a first plurality of sequestration pens disposedadjacent to each other such that each sequestration pen of the firstplurality opens off a first side of the microfluidic channel, and asecond plurality of sequestration pens disposed adjacent to each othersuch that each sequestration pen of the second plurality opens off asecond opposing side of the microfluidic channel; and a flowable polymersolution, wherein the polymer is capable of polymerization and/orthermally-induced gelling. In some embodiments, the microfluidic devicemay further include a barrier separating the first side of themicrofluidic channel from the second side of the microfluidic channel.In some embodiments, the barrier is punctuated by at least one gapaligned between a proximal opening to the microfluidic channel of afirst pen of the first plurality of pens and a proximal opening to themicrofluidic channel of a first pen of the second plurality of pens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an example of a system for use with a microfluidicdevice and associated control equipment according to some embodiments ofthe invention.

FIGS. 1B and 1C illustrate a microfluidic device according to someembodiments of the invention.

FIGS. 2A and 2B illustrate isolation pens according to some embodimentsof the invention.

FIG. 2C illustrates a detailed sequestration pen according to someembodiments of the invention.

FIGS. 2D-F illustrate sequestration pens according to some otherembodiments of the invention.

FIG. 2G illustrates a microfluidic device according to an embodiment ofthe invention.

FIG. 2H illustrates a coated surface of the microfluidic deviceaccording to an embodiment of the invention.

FIG. 3A illustrates a specific example of a system for use with amicrofluidic device and associated control equipment according to someembodiments of the invention.

FIG. 3B illustrates an imaging device according to some embodiments ofthe invention.

FIG. 4 is a photographic representation of one embodiment of insitu-generated isolation structures.

FIGS. 5A and 5B are photographic representations of another embodimentof in situ-generated isolation structures within a microfluidic (ornanofluidic) channel.

FIG. 6 is a graphical representation of another embodiment of an insitu-generated isolation structure in a microfluidic (or nanofluidic)channel.

FIG. 7 is a graphical representation of another embodiment of an insitu-generated isolation structure in a microfluidic (or nanofluidic)channel.

FIGS. 8A and 8B are graphical representations of another embodiment ofan in situ-generated isolation structure in a microfluidic (ornanofluidic) channel located at the proximal opening of an isolationpen.

FIGS. 9A-D are graphical representations of another embodiment of insitu-generated isolation structures within isolation pens.

FIGS. 10A-C are graphical representations of another embodiment of insitu-generated isolation structures within a microfluidic (ornanofluidic) flow region.

FIGS. 11A and 11B are graphical representations of another embodiment ofan in situ-generated isolation structure within an isolation pen.

FIGS. 12A and 12B are graphical representations of another embodiment ofin situ-generated isolation structures within a microfluidic (ornanofluidic) flow region.

FIG. 13A-C are graphical representations of another embodiment of insitu-generated isolation structures within microfluidic channels in aflow region of a microfluidic (or nanofluidic) device.

FIGS. 14A and 14B are graphical representation of another embodiment ofan in situ-generated isolation structure within a microfluidic (ornanofluidic) channel.

FIG. 15 is a graphical representation of another embodiment of an insitu-generated isolation structure within a microfluidic (ornanofluidic) channel.

FIG. 16 is a graphical representation of another embodiment of an insitu-generated isolation structure within an isolation pen

FIG. 17 is a graphical representation of another embodiment of insitu-generated isolation structures within a microfluidic (ornanofluidic) flow region.

FIG. 18 is a photographic representation of a rapidly prototypedmicrofluidic (or nanofluidic) enclosure using in situ-generatedisolation structures.

FIGS. 19A and 19B are graphical representations of other embodiments ofin situ-generated isolation structures within microfluidic (ornanofluidic) flow regions.

FIGS. 20A and 20B are graphical representations of other embodiments ofin situ-generated isolation structures within isolation pens of amicrofluidic (or nanofluidic) device.

FIGS. 21A-C are photographic representations of in situ-generatedisolation structures of a microfluidic (or nanofluidic) device,according to an embodiment of the invention.

FIG. 22 is a photographic representation of an embodiment of insitu-generated isolation structures within sequestration pens of amicrofluidic (or nanofluidic) device.

DETAILED DESCRIPTION OF THE INVENTION

This specification describes exemplary embodiments and applications ofthe invention. The invention, however, is not limited to these exemplaryembodiments and applications or to the manner in which the exemplaryembodiments and applications operate or are described herein. Moreover,the Figures may show simplified or partial views, and the dimensions ofelements in the Figures may be exaggerated or otherwise not inproportion for clarity. In addition, as the terms “on,” “attached to,”or “coupled to” are used herein, one element (e.g., a material, a layer,a substrate, etc.) can be “on,” “attached to,” or “coupled to” anotherelement regardless of whether the one element is directly on, attached,or coupled to the other element or there are one or more interveningelements between the one element and the other element. Also, directions(e.g., above, below, top, bottom, side, up, down, under, over, upper,lower, horizontal, vertical, “x,” “y,” “z,” etc.), if provided, arerelative and provided solely by way of example and for ease ofillustration and discussion and not by way of limitation. In addition,where reference is made to a list of elements (e.g., elements a, b, c),such reference is intended to include any one of the listed elements byitself, any combination of less than all of the listed elements, and/ora combination of all of the listed elements.

Section divisions in the specification are for ease of review only anddo not limit any combination of elements discussed.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “substantially” thus allows for minor,insignificant variations from an absolute or perfect state, dimension,measurement, result, or the like such as would be expected by a personof ordinary skill in the field but that do not appreciably affectoverall performance. When used with respect to numerical values orparameters or characteristics that can be expressed as numerical values,“substantially” means within ten percent.

As used herein, the term “ones” means more than one. As used herein, theterm “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

As used herein: μm means micrometer, μm³ means cubic micrometer, pLmeans picoliter, nL means nanoliter, and μL (or uL) means microliter.

As used herein, the term “transparent” refers to a material which allowsvisible light to pass through without substantially altering the lightas is passes through.

As used herein, the term “disposed” encompasses within its meaning“located.”

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is atype of microfluidic device having a microfluidic circuit that containsat least one circuit element, including but not limited to region(s),flow path(s), channel(s), chamber(s), and/or pen(s), configured to holda volume of fluid of less than about 1 μL, e.g., less than about 750,500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2,1 nL or less. A nanofluidic device may comprise a plurality of circuitelements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, ormore). In certain embodiments, one or more (e.g., all) of the at leastone circuit elements is configured to hold a volume of fluid of about100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pLto 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, oneor more (e.g., all) of the at least one circuit elements is configuredto hold a volume of fluid of about 20 nL to 200 nL, 100 to 200 nL, 100to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL,200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500nL, 250 to 600 nL, or 250 to 750 nL.

As used herein, a “flow path” refers to one or more fluidicallyconnected circuit elements (e.g. channel(s), region(s), chamber(s) andthe like) that define, and are subject to, the trajectory of a flow ofmedium. A flow path is thus an example of a swept region of amicrofluidic device. Other circuit elements (e.g., unswept regions) maybe fluidically connected with the circuit elements that comprise theflow path without being subject to the flow of medium in the flow path.

A “microfluidic channel” or “flow channel” as used herein refers to flowregion of a microfluidic device having a length that is significantlylonger than both the horizontal and vertical dimensions. For example,the flow channel can be at least 5 times the length of either thehorizontal or vertical dimension, e.g., at least 10 times the length, atleast 25 times the length, at least 100 times the length, at least 200times the length, at least 500 times the length, at least 1,000 timesthe length, at least 5,000 times the length, or longer. In someembodiments, the length of a flow channel is in the range of from about100,000 microns to about 500,000 microns, including any rangetherebetween. In some embodiments, the horizontal dimension is in therange of from about 100 microns to about 1000 microns (e.g., about 150to about 500 microns) and the vertical dimension is in the range of fromabout 25 microns to about 200 microns, e.g., from about 40 to about 150microns. It is noted that a flow channel may have a variety of differentspatial configurations in a microfluidic device, and thus is notrestricted to a perfectly linear element. For example, a flow channelmay be, or include one or more sections having, the followingconfigurations: curve, bend, spiral, incline, decline, fork (e.g.,multiple different flow paths), and any combination thereof. Inaddition, a flow channel may have different cross-sectional areas alongits path, widening and constricting to provide a desired fluid flowtherein.

As used herein, the term “obstruction” refers generally to a bump orsimilar type of structure that is sufficiently large so as to partially(but not completely) impede movement of target micro-objects between twodifferent regions or circuit elements in a microfluidic device. The twodifferent regions/circuit elements can be, for example, the connectionregion and the isolation region of a microfluidic sequestration pen.

As used herein, the term “constriction” refers generally to a narrowingof a width of a circuit element (or an interface between two circuitelements) in a microfluidic device. The constriction can be located, forexample, at the interface between the isolation region and theconnection region of a microfluidic sequestration pen of the instantinvention.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that, averaged over time, is less than the rate of diffusion ofcomponents of a material (e.g., an analyte of interest) into or withinthe fluidic medium. The rate of diffusion of components of such amaterial can depend on, for example, temperature, the size of thecomponents, and the strength of interactions between the components andthe fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through thedevice.

A microfluidic (or nanofluidic) device can comprise “swept” regions and“unswept” regions. As used herein, a “swept” region is comprised of oneor more fluidically interconnected circuit elements of a microfluidiccircuit, each of which experiences a flow of medium when fluid isflowing through the microfluidic circuit. The circuit elements of aswept region can include, for example, regions, channels, and all orparts of chambers. As used herein, an “unswept” region is comprised ofone or more fluidically interconnected circuit element of a microfluidiccircuit, each of which experiences substantially no flux of fluid whenfluid is flowing through the microfluidic circuit. An unswept region canbe fluidically connected to a swept region, provided the fluidicconnections are structured to enable diffusion but substantially no flowof media between the swept region and the unswept region. Themicrofluidic device can thus be structured to substantially isolate anunswept region from a flow of medium in a swept region, while enablingsubstantially only diffusive fluidic communication between the sweptregion and the unswept region. For example, a flow channel of amicro-fluidic device is an example of a swept region while an isolationregion (described in further detail below) of a microfluidic device isan example of an unswept region.

The capability of biological micro-objects (e.g., biological cells) toproduce specific biological materials (e.g., proteins, such asantibodies) can be assayed in such a microfluidic device. In a specificembodiment of an assay, sample material comprising biologicalmicro-objects (e.g., cells) to be assayed for production of an analyteof interest can be loaded into a swept region of the microfluidicdevice. Ones of the biological micro-objects (e.g., mammalian cells,such as human cells) can be selected for particular characteristics anddisposed in unswept regions. The remaining sample material can then beflowed out of the swept region and an assay material flowed into theswept region. Because the selected biological micro-objects are inunswept regions, the selected biological micro-objects are notsubstantially affected by the flowing out of the remaining samplematerial or the flowing in of the assay material. The selectedbiological micro-objects can be allowed to produce the analyte ofinterest, which can diffuse from the unswept regions into the sweptregion, where the analyte of interest can react with the assay materialto produce localized detectable reactions, each of which can becorrelated to a particular unswept region. Any unswept region associatedwith a detected reaction can be analyzed to determine which, if any, ofthe biological micro-objects in the unswept region are sufficientproducers of the analyte of interest.

As used herein, the term “micro-object” refers generally to anymicroscopic object that may be isolated and/or manipulated in accordancewith the present invention. Non-limiting examples of micro-objectsinclude: inanimate micro-objects such as microparticles; microbeads(e.g., polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells; biological organelles; vesicles, orcomplexes; synthetic vesicles; liposomes (e.g., synthetic or derivedfrom membrane preparations); lipid nanorafts, and the like; or acombination of inanimate micro-objects and biological micro-objects(e.g., microbeads attached to cells, liposome-coated micro-beads,liposome-coated magnetic beads, or the like). Beads may includemoieties/molecules covalently or non-covalently attached, such asfluorescent labels, proteins, carbohydrates, antigens, small moleculesignaling moieties, or other chemical/biological species capable of usein an assay. Lipid nanorafts have been described, for example, inRitchie et al. (2009) “Reconstitution of Membrane Proteins inPhospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231.

As used herein, the term “cell” is used interchangeably with the term“biological cell.” Non-limiting examples of biological cells includeeukaryotic cells, plant cells, animal cells, such as mammalian cells,reptilian cells, avian cells, fish cells, or the like, prokaryoticcells, bacterial cells, fungal cells, protozoan cells, or the like,cells dissociated from a tissue, such as muscle, cartilage, fat, skin,liver, lung, neural tissue, and the like, immunological cells, such as Tcells, B cells, natural killer cells, macrophages, and the like, embryos(e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells,cells from a cell line, cancer cells, infected cells, transfected and/ortransformed cells, reporter cells, and the like. A mammalian cell canbe, for example, from a human, a mouse, a rat, a horse, a goat, a sheep,a cow, a primate, or the like.

A colony of biological cells is “clonal” if all of the living cells inthe colony that are capable of reproducing are daughter cells derivedfrom a single parent cell. In certain embodiments, all the daughtercells in a clonal colony are derived from the single parent cell by nomore than 10 divisions. In other embodiments, all the daughter cells ina clonal colony are derived from the single parent cell by no more than14 divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 17divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 20divisions. The term “clonal cells” refers to cells of the same clonalcolony.

As used herein, a “colony” of biological cells refers to 2 or more cells(e.g. about 2 to about 20, about 4 to about 40, about 6 to about 60,about 8 to about 80, about 10 to about 100, about 20 about 200, about 40about 400, about 60 about 600, about 80 about 800, about 100 about 1000,or greater than 1000 cells).

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components and,optionally a surface, that provides the conditions necessary to keep thecells viable and/or expanding.

As used herein, the term “expanding” when referring to cells, refers toincreasing in cell number.

As used herein, the term “processing” when referring to processingcells, may include culturing or continuing to culture the cells,assaying the cells using one or more assays, and/or preparing the cellsfor a procedure such as, but not limited to, lysis, fusion,transfection, gene editing of any kind (e.g., targeted gene editing)and/or genotyping.

As used herein, “isolating a micro-object” means confining amicro-object to a defined area within the microfluidic device. Themicro-object may still be capable of motion within the defined area(e.g., within an in situ-generated isolation structure).

As used herein, “antibody” refers to an immunoglobulin (Ig) and includesboth polyclonal and monoclonal antibodies; primatized (e.g., humanized);murine; mouse-human; mouse-primate; and chimeric; and may be an intactmolecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab′ andF(ab)′2 fragments), or multimers or aggregates of intact moleculesand/or fragments; and may occur in nature or be produced, e.g., byimmunization, synthesis or genetic engineering. An “antibody fragment,”as used herein, refers to fragments, derived from or related to anantibody, which bind antigen and which in some embodiments may bederivatized to exhibit structural features that facilitate clearance anduptake, e.g., by the incorporation of galactose residues. This includes,e.g., F(ab), F(ab)′2, scFv, light chain variable region (VL), heavychain variable region (VH), and combinations thereof.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein, “capture moiety” is a chemical or biological species,functionality, or motif that provides a recognition site for amicro-object. A selected class of micro-objects may recognize thecapture moiety and may bind or have an affinity for the capture moiety.Non-limiting examples include antigens, antibodies, and cell surfacebinding motifs.

As used herein, “flowable polymer” is a polymer monomer or macromer thatis soluble or dispersible within a fluidic medium. The flowable polymermay be input into a microfluidic flow region and flow with othercomponents of a fluidic medium therein.

As used herein, “photoinitiated polymer” refers to a polymer (or amonomeric molecule that can be used to generate the polymer) that uponexposure to light, is capable of crosslinking covalently, formingspecific covalent bonds, changing regiochemistry around a rigidifiedchemical motif, or forming ion pairs which cause a change in physicalstate, and thereby forming a polymer network. In some instances, aphotoinitiated polymer may include a polymer segment bound to one ormore chemical moieties capable of crosslinking covalently, formingspecific covalent bonds, changing regiochemistry around a rigidifiedchemical motif, or forming ion pairs which cause a change in physicalstate. In some instances, a photoinitiated polymer may require aphotoactivatable radical initiator to initiate formation of the polymernetwork (e.g., via polymerization of the polymer).

Microfluidic Devices and Systems for Operating and Observing SuchDevices.

FIG. 1A illustrates an example of a microfluidic device 100 and a system150 which can be used for generation of embryos in vitro, includingselecting and evaluating ova and/or oocytes and/or sperm. A perspectiveview of the microfluidic device 100 is shown having a partial cut-awayof its cover 110 to provide a partial view into the microfluidic device100. The microfluidic device 100 generally comprises a microfluidiccircuit 120 comprising a flow path 106 through which a fluidic medium180 can flow, optionally carrying one or more micro-objects (not shown)into and/or through the microfluidic circuit 120. Although a singlemicrofluidic circuit 120 is illustrated in FIG. 1A, suitablemicrofluidic devices can include a plurality (e.g., 2 or 3) of suchmicrofluidic circuits. Regardless, the microfluidic device 100 can beconfigured to be a nanofluidic device. As illustrated in FIG. 1A, themicrofluidic circuit 120 may include a plurality of microfluidicsequestration pens 124, 126, 128, and 130, where each sequestration pensmay have one or more openings in fluidic communication with flow path106. In some embodiments of the device of FIG. 1A, the sequestrationpens may have only a single opening in fluidic communication with theflow path 106. As discussed further below, the microfluidicsequestration pens comprise various features and structures that havebeen optimized for retaining micro-objects in the microfluidic device,such as microfluidic device 100, even when a medium 180 is flowingthrough the flow path 106. Before turning to the foregoing, however, abrief description of microfluidic device 100 and system 150 is provided.

As generally illustrated in FIG. 1A, the microfluidic circuit 120 isdefined by an enclosure 102. Although the enclosure 102 can bephysically structured in different configurations, in the example shownin FIG. 1A the enclosure 102 is depicted as comprising a supportstructure 104 (e.g., a base), a microfluidic circuit structure 108, anda cover 110. The support structure 104, microfluidic circuit structure108, and cover 110 can be attached to each other. For example, themicrofluidic circuit structure 108 can be disposed on an inner surface109 of the support structure 104, and the cover 110 can be disposed overthe microfluidic circuit structure 108. Together with the supportstructure 104 and cover 110, the microfluidic circuit structure 108 candefine the elements of the microfluidic circuit 120.

The support structure 104 can be at the bottom and the cover 110 at thetop of the microfluidic circuit 120 as illustrated in FIG. 1A.Alternatively, the support structure 104 and the cover 110 can beconfigured in other orientations. For example, the support structure 104can be at the top and the cover 110 at the bottom of the microfluidiccircuit 120. Regardless, there can be one or more ports 107 eachcomprising a passage into or out of the enclosure 102. Examples of apassage include a valve, a gate, a pass-through hole, or the like. Asillustrated, port 107 is a pass-through hole created by a gap in themicrofluidic circuit structure 108. However, the port 107 can besituated in other components of the enclosure 102, such as the cover110. Only one port 107 is illustrated in FIG. 1A but the microfluidiccircuit 120 can have two or more ports 107. For example, there can be afirst port 107 that functions as an inlet for fluid entering themicrofluidic circuit 120, and there can be a second port 107 thatfunctions as an outlet for fluid exiting the microfluidic circuit 120.Whether a port 107 function as an inlet or an outlet can depend upon thedirection that fluid flows through flow path 106.

The support structure 104 can comprise one or more electrodes (notshown) and a substrate or a plurality of interconnected substrates. Forexample, the support structure 104 can comprise one or moresemiconductor substrates, each of which is electrically connected to anelectrode (e.g., all or a subset of the semiconductor substrates can beelectrically connected to a single electrode). The support structure 104can further comprise a printed circuit board assembly (“PCBA”). Forexample, the semiconductor substrate(s) can be mounted on a PCBA.

The microfluidic circuit structure 108 can define circuit elements ofthe microfluidic circuit 120. Such circuit elements can comprise spacesor regions that can be fluidly interconnected when microfluidic circuit120 is filled with fluid, such as flow regions (which may include or beone or more flow channels), chambers, pens, traps, and the like. In themicrofluidic circuit 120 illustrated in FIG. 1A, the microfluidiccircuit structure 108 comprises a frame 114 and a microfluidic circuitmaterial 116. The frame 114 can partially or completely enclose themicrofluidic circuit material 116. The frame 114 can be, for example, arelatively rigid structure substantially surrounding the microfluidiccircuit material 116. For example, the frame 114 can comprise a metalmaterial.

The microfluidic circuit material 116 can be patterned with cavities orthe like to define circuit elements and interconnections of themicrofluidic circuit 120. The microfluidic circuit material 116 cancomprise a flexible material, such as a flexible polymer (e.g. rubber,plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or thelike), which can be gas permeable. Other examples of materials that cancompose microfluidic circuit material 116 include molded glass, anetchable material such as silicone (e.g. photo-patternable silicone or“PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, suchmaterials—and thus the microfluidic circuit material 116—can be rigidand/or substantially impermeable to gas. Regardless, microfluidiccircuit material 116 can be disposed on the support structure 104 andinside the frame 114.

The cover 110 can be an integral part of the frame 114 and/or themicrofluidic circuit material 116. Alternatively, the cover 110 can be astructurally distinct element, as illustrated in FIG. 1A. The cover 110can comprise the same or different materials than the frame 114 and/orthe microfluidic circuit material 116. Similarly, the support structure104 can be a separate structure from the frame 114 or microfluidiccircuit material 116 as illustrated, or an integral part of the frame114 or microfluidic circuit material 116. Likewise, the frame 114 andmicrofluidic circuit material 116 can be separate structures as shown inFIG. 1A or integral portions of the same structure.

In some embodiments, the cover 110 can comprise a rigid material. Therigid material may be glass or a material with similar properties. Insome embodiments, the cover 110 can comprise a deformable material. Thedeformable material can be a polymer, such as PDMS. In some embodiments,the cover 110 can comprise both rigid and deformable materials. Forexample, one or more portions of cover 110 (e.g., one or more portionspositioned over sequestration pens 124, 126, 128, 130) can comprise adeformable material that interfaces with rigid materials of the cover110. In some embodiments, the cover 110 can further include one or moreelectrodes. The one or more electrodes can comprise a conductive oxide,such as indium-tin-oxide (ITO), which may be coated on glass or asimilarly insulating material. Alternatively, the one or more electrodescan be flexible electrodes, such as single-walled nanotubes,multi-walled nanotubes, nanowires, clusters of electrically conductivenanoparticles, or combinations thereof, embedded in a deformablematerial, such as a polymer (e.g., PDMS). Flexible electrodes that canbe used in microfluidic devices have been described, for example, inU.S. 2012/0325665 (Chiou et al.), the contents of which are incorporatedherein by reference. In some embodiments, the cover 110 can be modified(e.g., by conditioning all or part of a surface that faces inward towardthe microfluidic circuit 120) to support cell adhesion, viability and/orgrowth. The modification may include a coating of a synthetic or naturalpolymer. In some embodiments, the cover 110 and/or the support structure104 can be transparent to light. The cover 110 may also include at leastone material that is gas permeable (e.g., PDMS or PPS).

FIG. 1A also shows a system 150 for operating and controllingmicrofluidic devices, such as microfluidic device 100. System 150includes an electrical power source 192, an imaging device 194(incorporated within imaging module 164, where device 194 is notillustrated in FIG. 1A, per se), and a tilting device 190 (part oftilting module 166, where device 190 is not illustrated in FIG. 1).

The electrical power source 192 can provide electric power to themicrofluidic device 100 and/or tilting device 190, providing biasingvoltages or currents as needed. The electrical power source 192 can, forexample, comprise one or more alternating current (AC) and/or directcurrent (DC) voltage or current sources. The imaging device 194 (part ofimaging module 164, discussed below) can comprise a device, such as adigital camera, for capturing images inside microfluidic circuit 120. Insome instances, the imaging device 194 further comprises a detectorhaving a fast frame rate and/or high sensitivity (e.g. for low lightapplications). The imaging device 194 can also include a mechanism fordirecting stimulating radiation and/or light beams into the microfluidiccircuit 120 and collecting radiation and/or light beams reflected oremitted from the microfluidic circuit 120 (or micro-objects containedtherein). The emitted light beams may be in the visible spectrum andmay, e.g., include fluorescent emissions. The reflected light beams mayinclude reflected emissions originating from an LED or a wide spectrumlamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or aXenon arc lamp. As discussed with respect to FIG. 3B, the imaging device194 may further include a microscope (or an optical train), which may ormay not include an eyepiece.

System 150 further comprises a tilting device 190 (part of tiltingmodule 166, discussed below) configured to rotate a microfluidic device100 about one or more axes of rotation. In some embodiments, the tiltingdevice 190 is configured to support and/or hold the enclosure 102comprising the microfluidic circuit 120 about at least one axis suchthat the microfluidic device 100 (and thus the microfluidic circuit 120)can be held in a level orientation (i.e. at 0° relative to x- andy-axes), a vertical orientation (i.e. at 90° relative to the x-axisand/or the y-axis), or any orientation therebetween. The orientation ofthe microfluidic device 100 (and the microfluidic circuit 120) relativeto an axis is referred to herein as the “tilt” of the microfluidicdevice 100 (and the microfluidic circuit 120). For example, the tiltingdevice 190 can tilt the microfluidic device 100 at 0.1°, 0.2°, 0.3°,0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°,25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relativeto the x-axis or any degree therebetween. The level orientation (andthus the x- and y-axes) is defined as normal to a vertical axis definedby the force of gravity. The tilting device can also tilt themicrofluidic device 100 (and the microfluidic circuit 120) to any degreegreater than 90° relative to the x-axis and/or y-axis, or tilt themicrofluidic device 100 (and the microfluidic circuit 120) 180° relativeto the x-axis or the y-axis in order to fully invert the microfluidicdevice 100 (and the microfluidic circuit 120). Similarly, in someembodiments, the tilting device 190 tilts the microfluidic device 100(and the microfluidic circuit 120) about an axis of rotation defined byflow path 106 or some other portion of microfluidic circuit 120.

In some instances, the microfluidic device 100 is tilted into a verticalorientation such that the flow path 106 is positioned above or below oneor more sequestration pens. The term “above” as used herein denotes thatthe flow path 106 is positioned higher than the one or moresequestration pens on a vertical axis defined by the force of gravity(i.e. an object in a sequestration pen above a flow path 106 would havea higher gravitational potential energy than an object in the flowpath). The term “below” as used herein denotes that the flow path 106 ispositioned lower than the one or more sequestration pens on a verticalaxis defined by the force of gravity (i.e. an object in a sequestrationpen below a flow path 106 would have a lower gravitational potentialenergy than an object in the flow path).

In some instances, the tilting device 190 tilts the microfluidic device100 about an axis that is parallel to the flow path 106. Moreover, themicrofluidic device 100 can be tilted to an angle of less than 90° suchthat the flow path 106 is located above or below one or moresequestration pens without being located directly above or below thesequestration pens. In other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis perpendicular to the flow path106. In still other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis that is neither parallel norperpendicular to the flow path 106.

System 150 can further include a media source 178. The media source 178(e.g., a container, reservoir, or the like) can comprise multiplesections or containers, each for holding a different fluidic medium 180.Thus, the media source 178 can be a device that is outside of andseparate from the microfluidic device 100, as illustrated in FIG. 1A.Alternatively, the media source 178 can be located in whole or in partinside the enclosure 102 of the microfluidic device 100. For example,the media source 178 can comprise reservoirs that are part of themicrofluidic device 100.

FIG. 1A also illustrates simplified block diagram depictions of examplesof control and monitoring equipment 152 that constitute part of system150 and can be utilized in conjunction with a microfluidic device 100.As shown, examples of such control and monitoring equipment 152 includea master controller 154 comprising a media module 160 for controllingthe media source 178, a motive module 162 for controlling movementand/or selection of micro-objects (not shown) and/or medium (e.g.,droplets of medium) in the microfluidic circuit 120, an imaging module164 for controlling an imaging device 194 (e.g., a camera, microscope,light source or any combination thereof) for capturing images (e.g.,digital images), and a tilting module 166 for controlling a tiltingdevice 190. The control equipment 152 can also include other modules 168for controlling, monitoring, or performing other functions with respectto the microfluidic device 100. As shown, the equipment 152 can furtherinclude a display device 170 and an input/output device 172.

The master controller 154 can comprise a control module 156 and adigital memory 158. The control module 156 can comprise, for example, adigital processor configured to operate in accordance with machineexecutable instructions (e.g., software, firmware, source code, or thelike) stored as non-transitory data or signals in the memory 158.Alternatively, or in addition, the control module 156 can comprisehardwired digital circuitry and/or analog circuitry. The media module160, motive module 162, imaging module 164, tilting module 166, and/orother modules 168 can be similarly configured. Thus, functions,processes acts, actions, or steps of a process discussed herein as beingperformed with respect to the microfluidic device 100 or any othermicrofluidic apparatus can be performed by any one or more of the mastercontroller 154, media module 160, motive module 162, imaging module 164,tilting module 166, and/or other modules 168 configured as discussedabove. Similarly, the master controller 154, media module 160, motivemodule 162, imaging module 164, tilting module 166, and/or other modules168 may be communicatively coupled to transmit and receive data used inany function, process, act, action or step discussed herein.

The media module 160 controls the media source 178. For example, themedia module 160 can control the media source 178 to input a selectedfluidic medium 180 into the enclosure 102 (e.g., through an inlet port107). The media module 160 can also control removal of media from theenclosure 102 (e.g., through an outlet port (not shown)). One or moremedia can thus be selectively input into and removed from themicrofluidic circuit 120. The media module 160 can also control the flowof fluidic medium 180 in the flow path 106 inside the microfluidiccircuit 120. For example, in some embodiments media module 160 stops theflow of media 180 in the flow path 106 and through the enclosure 102prior to the tilting module 166 causing the tilting device 190 to tiltthe microfluidic device 100 to a desired angle of incline.

The motive module 162 can be configured to control selection, trapping,and movement of micro-objects (not shown) in the microfluidic circuit120. As discussed below with respect to FIGS. 1B and 1C, the enclosure102 can comprise a dielectrophoresis (DEP), optoelectronic tweezers(OET) and/or opto-electrowetting (OEW) configuration (not shown in FIG.1A), and the motive module 162 can control the activation of electrodesand/or transistors (e.g., phototransistors) to select and movemicro-objects (not shown) and/or droplets of medium (not shown) in theflow path 106 and/or sequestration pens 124, 126, 128, 130.

The imaging module 164 can control the imaging device 194. For example,the imaging module 164 can receive and process image data from theimaging device 194. Image data from the imaging device 194 can compriseany type of information captured by the imaging device 194 (e.g., thepresence or absence of micro-objects, droplets of medium, accumulationof label, such as fluorescent label, etc.). Using the informationcaptured by the imaging device 194, the imaging module 164 can furthercalculate the position of objects (e.g., micro-objects, droplets ofmedium) and/or the rate of motion of such objects within themicrofluidic device 100.

The tilting module 166 can control the tilting motions of tilting device190. Alternatively, or in addition, the tilting module 166 can controlthe tilting rate and timing to optimize transfer of micro-objects to theone or more sequestration pens via gravitational forces. The tiltingmodule 166 is communicatively coupled with the imaging module 164 toreceive data describing the motion of micro-objects and/or droplets ofmedium in the microfluidic circuit 120. Using this data, the tiltingmodule 166 may adjust the tilt of the microfluidic circuit 120 in orderto adjust the rate at which micro-objects and/or droplets of medium movein the microfluidic circuit 120. The tilting module 166 may also usethis data to iteratively adjust the position of a micro-object and/ordroplet of medium in the microfluidic circuit 120.

In the example shown in FIG. 1A, the microfluidic circuit 120 isillustrated as comprising a microfluidic channel 122 and sequestrationpens 124, 126, 128, 130. Each pen comprises an opening to channel 122,but otherwise is enclosed such that the pens can substantially isolatemicro-objects inside the pen from fluidic medium 180 and/ormicro-objects in the flow path 106 of channel 122 or in other pens. Thewalls of the sequestration pen extend from the inner surface 109 of thebase to the inside surface of the cover 110 to provide enclosure. Theopening of the pen to the microfluidic channel 122 is oriented at anangle to the flow 106 of fluidic medium 180 such that flow 106 is notdirected into the pens. The flow may be tangential or orthogonal to theplane of the opening of the pen. In some instances, pens 124, 126, 128,130 are configured to physically corral one or more micro-objects withinthe microfluidic circuit 120. Sequestration pens in accordance with thepresent invention can comprise various shapes, surfaces and featuresthat are optimized for use with DEP, OET, OEW, fluid flow, and/orgravitational forces, as will be discussed and shown in detail below.

The microfluidic circuit 120 may comprise any number of microfluidicsequestration pens. Although five sequestration pens are shown,microfluidic circuit 120 may have fewer or more sequestration pens. Asshown, microfluidic sequestration pens 124, 126, 128, and 130 ofmicrofluidic circuit 120 each comprise differing features and shapeswhich may provide one or more benefits useful in producing an embryo,such as isolating one ovum from an adjacent ovum. Testing, stimulatingand fertilizing may all be performed on an individual basis and, in someembodiments, may be performed on an individual time scale. In someembodiments, the microfluidic circuit 120 comprises a plurality ofidentical microfluidic sequestration pens.

In some embodiments, the microfluidic circuit 120 comprises a pluralityof microfluidic sequestration pens, wherein two or more of thesequestration pens comprise differing structures and/or features whichprovide differing benefits in producing embryos. One non-limitingexample may include maintaining ova in one type of pen while maintainingsperm in a different type of pen. In another embodiment, at least one ofthe sequestration pens is configured to have electrical contactssuitable for providing electrical activation for an ovum. In yet anotherembodiment, differing types of cells (such as, for example, uterinecells, endometrial cells, PEG (intercalary) cells derived from theuterine tube (e.g., oviduct or Fallopian tube), cumulus cells, or acombination thereof) may be disposed in sequestration pens adjacent to asequestration pen containing an ovum, such that secretions from thesurrounding sequestration pens may diffuse out of each respective penand into the pen containing an ovum, which is not possible withmacroscale in-vitro culturing and fertilization. Microfluidic devicesuseful for producing an embryo may include any of the sequestration pens124, 126, 128, and 130 or variations thereof, and/or may include pensconfigured like those shown in FIGS. 2B, 2C, 2D, 2E and 2F, as discussedbelow.

In the embodiment illustrated in FIG. 1A, a single channel 122 and flowpath 106 is shown. However, other embodiments may contain multiplechannels 122, each configured to comprise a flow path 106. Themicrofluidic circuit 120 further comprises an inlet valve or port 107 influid communication with the flow path 106 and fluidic medium 180,whereby fluidic medium 180 can access channel 122 via the inlet port107. In some instances, the flow path 106 comprises a single path. Insome instances, the single path is arranged in a zigzag pattern wherebythe flow path 106 travels across the microfluidic device 100 two or moretimes in alternating directions.

In some instances, microfluidic circuit 120 comprises a plurality ofparallel channels 122 and flow paths 106, wherein the fluidic medium 180within each flow path 106 flows in the same direction. In someinstances, the fluidic medium within each flow path 106 flows in atleast one of a forward or reverse direction. In some instances, aplurality of sequestration pens is configured (e.g., relative to achannel 122) such that the sequestration pens can be loaded with targetmicro-objects in parallel.

In some embodiments, microfluidic circuit 120 further comprises one ormore micro-object traps 132. The traps 132 are generally formed in awall forming the boundary of a channel 122, and may be positionedopposite an opening of one or more of the microfluidic sequestrationpens 124, 126, 128, 130. In some embodiments, the traps 132 areconfigured to receive or capture a single micro-object from the flowpath 106. In some embodiments, the traps 132 are configured to receiveor capture a plurality of micro-objects from the flow path 106. In someinstances, the traps 132 comprise a volume approximately equal to thevolume of a single target micro-object.

The traps 132 may further comprise an opening which is configured toassist the flow of targeted micro-objects into the traps 132. In someinstances, the traps 132 comprise an opening having a height and widththat is approximately equal to the dimensions of a single targetmicro-object, whereby larger micro-objects are prevented from enteringinto the micro-object trap. The traps 132 may further comprise otherfeatures configured to assist in retention of targeted micro-objectswithin the trap 132. In some instances, the trap 132 is aligned with andsituated on the opposite side of a channel 122 relative to the openingof a microfluidic sequestration pen, such that upon tilting themicrofluidic device 100 about an axis parallel to the channel 122, thetrapped micro-object exits the trap 132 at a trajectory that causes themicro-object to fall into the opening of the sequestration pen. In someinstances, the trap 132 comprises a side passage 134 that is smallerthan the target micro-object in order to facilitate flow through thetrap 132 and thereby increase the likelihood of capturing a micro-objectin the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied acrossthe fluidic medium 180 (e.g., in the flow path and/or in thesequestration pens) via one or more electrodes (not shown) tomanipulate, transport, separate and sort micro-objects located therein.For example, in some embodiments, DEP forces are applied to one or moreportions of microfluidic circuit 120 in order to transfer a singlemicro-object from the flow path 106 into a desired microfluidicsequestration pen. In some embodiments, DEP forces are used to prevent amicro-object within a sequestration pen (e.g., sequestration pen 124,126, 128, or 130) from being displaced therefrom. Further, in someembodiments, DEP forces are used to selectively remove a micro-objectfrom a sequestration pen that was previously collected in accordancewith the teachings of the instant invention. In some embodiments, theDEP forces comprise optoelectronic tweezer (OET) forces.

In other embodiments, optoelectrowetting (OEW) forces are applied to oneor more positions in the support structure 104 (and/or the cover 110) ofthe microfluidic device 100 (e.g., positions helping to define the flowpath and/or the sequestration pens) via one or more electrodes (notshown) to manipulate, transport, separate and sort droplets located inthe microfluidic circuit 120. For example, in some embodiments, OEWforces are applied to one or more positions in the support structure 104(and/or the cover 110) in order to transfer a single droplet from theflow path 106 into a desired microfluidic sequestration pen. In someembodiments, OEW forces are used to prevent a droplet within asequestration pen (e.g., sequestration pen 124, 126, 128, or 130) frombeing displaced therefrom. Further, in some embodiments, OEW forces areused to selectively remove a droplet from a sequestration pen that waspreviously collected in accordance with the teachings of the instantinvention.

In some embodiments, DEP and/or OEW forces are combined with otherforces, such as flow and/or gravitational force, so as to manipulate,transport, separate and sort micro-objects and/or droplets within themicrofluidic circuit 120. For example, the enclosure 102 can be tilted(e.g., by tilting device 190) to position the flow path 106 andmicro-objects located therein above the microfluidic sequestration pens,and the force of gravity can transport the micro-objects and/or dropletsinto the pens. In some embodiments, the DEP and/or OEW forces can beapplied prior to the other forces. In other embodiments, the DEP and/orOEW forces can be applied after the other forces. In still otherinstances, the DEP and/or OEW forces can be applied at the same time asthe other forces or in an alternating manner with the other forces.

FIGS. 1B, 1C, and 2A-2H illustrates various embodiments of microfluidicdevices that can be used in the practice of the present invention. FIG.1B depicts an embodiment in which the microfluidic device 200 isconfigured as an optically-actuated electrokinetic device. A variety ofoptically-actuated electrokinetic devices are known in the art,including devices having an optoelectronic tweezer (OET) configurationand devices having an opto-electrowetting (OEW) configuration. Examplesof suitable OET configurations are illustrated in the following U.S.patent documents, each of which is incorporated herein by reference inits entirety: U.S. Pat. No. RE 44,711 (Wu et al.) (originally issued asU.S. Pat. No. 7,612,355); and U.S. Pat. No. 7,956,339 (Ohta et al.).Examples of OEW configurations are illustrated in U.S. Pat. No.6,958,132 (Chiou et al.) and U.S. Patent Application Publication No.2012/0024708 (Chiou et al.), both of which are incorporated by referenceherein in their entirety. Yet another example of an optically-actuatedelectrokinetic device includes a combined OET/OEW configuration,examples of which are shown in U.S. Patent Publication Nos. 20150306598(Khandros et al.) and 20150306599 (Khandros et al.) and theircorresponding PCT Publications WO2015/164846 and WO2015/164847, all ofwhich are incorporated herein by reference in their entirety.

Examples of microfluidic devices having pens in which oocytes, ova, orembryos can be placed, cultured, and/or monitored have been described,for example, in US 2014/0116881 (application Ser. No. 14/060,117, filedOct. 22, 2013), US 2015/0151298 (application Ser. No. 14/520,568, filedOct. 22, 2014), and US 2015/0165436 (application Ser. No. 14/521,447,filed Oct. 22, 2014), each of which is incorporated herein by referencein its entirety. U.S. application Ser. Nos. 14/520,568 and 14/521,447also describe exemplary methods of analyzing secretions of cellscultured in a microfluidic device. Each of the foregoing applicationsfurther describes microfluidic devices configured to producedielectrophoretic (DEP) forces, such as optoelectronic tweezers (OET) orconfigured to provide opto-electro wetting (OEW). For example, theoptoelectronic tweezers device illustrated in FIG. 2 of US 2014/0116881is an example of a device that can be utilized in embodiments of thepresent invention to select and move an individual biologicalmicro-object or a group of biological micro-objects.

Microfluidic Device Motive Configurations.

As described above, the control and monitoring equipment of the systemcan comprise a motive module for selecting and moving objects, such asmicro-objects or droplets, in the microfluidic circuit of a microfluidicdevice. The microfluidic device can have a variety of motiveconfigurations, depending upon the type of object being moved and otherconsiderations. For example, a dielectrophoresis (DEP) configuration canbe utilized to select and move micro-objects in the microfluidiccircuit. Thus, the support structure 104 and/or cover 110 of themicrofluidic device 100 can comprise a DEP configuration for selectivelyinducing DEP forces on micro-objects in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual micro-objects or groups of micro-objects. Alternatively, thesupport structure 104 and/or cover 110 of the microfluidic device 100can comprise an electrowetting (EW) configuration for selectivelyinducing EW forces on droplets in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual droplets or groups of droplets.

One example of a microfluidic device 200 comprising a DEP configurationis illustrated in FIGS. 1B and 1C. While for purposes of simplicityFIGS. 1B and 1C show a side cross-sectional view and a topcross-sectional view, respectively, of a portion of an enclosure 102 ofthe microfluidic device 200 having an open region/chamber 202, it shouldbe understood that the region/chamber 202 may be part of a fluidiccircuit element having a more detailed structure, such as a growthchamber, a sequestration pen, a flow region, or a flow channel.Furthermore, the microfluidic device 200 may include other fluidiccircuit elements. For example, the microfluidic device 200 can include aplurality of growth chambers or sequestration pens and/or one or moreflow regions or flow channels, such as those described herein withrespect to microfluidic device 100. A DEP configuration may beincorporated into any such fluidic circuit elements of the microfluidicdevice 200, or select portions thereof. It should be further appreciatedthat any of the above or below described microfluidic device componentsand system components may be incorporated in and/or used in combinationwith the microfluidic device 200. For example, system 150 includingcontrol and monitoring equipment 152, described above, may be used withmicrofluidic device 200, including one or more of the media module 160,motive module 162, imaging module 164, tilting module 166, and othermodules 168.

As seen in FIG. 1B, the microfluidic device 200 includes a supportstructure 104 having a bottom electrode 204 and an electrode activationsubstrate 206 overlying the bottom electrode 204, and a cover 110 havinga top electrode 210, with the top electrode 210 spaced apart from thebottom electrode 204. The top electrode 210 and the electrode activationsubstrate 206 define opposing surfaces of the region/chamber 202. Amedium 180 contained in the region/chamber 202 thus provides a resistiveconnection between the top electrode 210 and the electrode activationsubstrate 206. A power source 212 configured to be connected to thebottom electrode 204 and the top electrode 210 and create a biasingvoltage between the electrodes, as required for the generation of DEPforces in the region/chamber 202, is also shown. The power source 212can be, for example, an alternating current (AC) power source.

In certain embodiments, the microfluidic device 200 illustrated in FIGS.1B and 1C can have an optically-actuated DEP configuration. Accordingly,changing patterns of light 218 from the light source 216, which may becontrolled by the motive module 162, can selectively activate anddeactivate changing patterns of DEP electrodes at regions 214 of theinner surface 208 of the electrode activation substrate 206.(Hereinafter the regions 214 of a microfluidic device having a DEPconfiguration are referred to as “DEP electrode regions.”) Asillustrated in FIG. 1C, a light pattern 218 directed onto the innersurface 208 of the electrode activation substrate 206 can illuminateselect DEP electrode regions 214 a (shown in white) in a pattern, suchas a square. The non-illuminated DEP electrode regions 214(cross-hatched) are hereinafter referred to as “dark” DEP electroderegions 214. The relative electrical impedance through the DEP electrodeactivation substrate 206 (i.e., from the bottom electrode 204 up to theinner surface 208 of the electrode activation substrate 206 whichinterfaces with the medium 180 in the flow region 106) is greater thanthe relative electrical impedance through the medium 180 in theregion/chamber 202 (i.e., from the inner surface 208 of the electrodeactivation substrate 206 to the top electrode 210 of the cover 110) ateach dark DEP electrode region 214. An illuminated DEP electrode region214 a, however, exhibits a reduced relative impedance through theelectrode activation substrate 206 that is less than the relativeimpedance through the medium 180 in the region/chamber 202 at eachilluminated DEP electrode region 214 a.

With the power source 212 activated, the foregoing DEP configurationcreates an electric field gradient in the fluidic medium 180 betweenilluminated DEP electrode regions 214 a and adjacent dark DEP electroderegions 214, which in turn creates local DEP forces that attract orrepel nearby micro-objects (not shown) in the fluidic medium 180. DEPelectrodes that attract or repel micro-objects in the fluidic medium 180can thus be selectively activated and deactivated at many different suchDEP electrode regions 214 at the inner surface 208 of the region/chamber202 by changing light patterns 218 projected from a light source 216into the microfluidic device 200. Whether the DEP forces attract orrepel nearby micro-objects can depend on such parameters as thefrequency of the power source 212 and the dielectric properties of themedium 180 and/or micro-objects (not shown).

The square pattern 220 of illuminated DEP electrode regions 214 aillustrated in FIG. 1C is an example only. Any pattern of the DEPelectrode regions 214 can be illuminated (and thereby activated) by thepattern of light 218 projected into the device 200, and the pattern ofilluminated/activated DEP electrode regions 214 can be repeatedlychanged by changing or moving the light pattern 218.

In some embodiments, the electrode activation substrate 206 can compriseor consist of a photoconductive material. In such embodiments, the innersurface 208 of the electrode activation substrate 206 can befeatureless. For example, the electrode activation substrate 206 cancomprise or consist of a layer of hydrogenated amorphous silicon(a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen(calculated as 100*the number of hydrogen atoms/the total number ofhydrogen and silicon atoms). The layer of a-Si:H can have a thickness ofabout 500 nm to about 2.0 μm. In such embodiments, the DEP electroderegions 214 can be created anywhere and in any pattern on the innersurface 208 of the electrode activation substrate 206, in accordancewith the light pattern 218. The number and pattern of the DEP electroderegions 214 thus need not be fixed, but can correspond to the lightpattern 218. Examples of microfluidic devices having a DEP configurationcomprising a photoconductive layer such as discussed above have beendescribed, for example, in U.S. Pat. No. RE 44,711 (Wu et al.)(originally issued as U.S. Pat. No. 7,612,355), the entire contents ofwhich are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 cancomprise a substrate comprising a plurality of doped layers,electrically insulating layers (or regions), and electrically conductivelayers that form semiconductor integrated circuits, such as is known insemiconductor fields. For example, the electrode activation substrate206 can comprise a plurality of phototransistors, including, forexample, lateral bipolar phototransistors, each phototransistorcorresponding to a DEP electrode region 214. Alternatively, theelectrode activation substrate 206 can comprise electrodes (e.g.,conductive metal electrodes) controlled by phototransistor switches,with each such electrode corresponding to a DEP electrode region 214.The electrode activation substrate 206 can include a pattern of suchphototransistors or phototransistor-controlled electrodes. The pattern,for example, can be an array of substantially square phototransistors orphototransistor-controlled electrodes arranged in rows and columns, suchas shown in FIG. 2B. Alternatively, the pattern can be an array ofsubstantially hexagonal phototransistors or phototransistor-controlledelectrodes that form a hexagonal lattice. Regardless of the pattern,electric circuit elements can form electrical connections between theDEP electrode regions 214 at the inner surface 208 of the electrodeactivation substrate 206 and the bottom electrode 210, and thoseelectrical connections (i.e., phototransistors or electrodes) can beselectively activated and deactivated by the light pattern 218. When notactivated, each electrical connection can have high impedance such thatthe relative impedance through the electrode activation substrate 206(i.e., from the bottom electrode 204 to the inner surface 208 of theelectrode activation substrate 206 which interfaces with the medium 180in the region/chamber 202) is greater than the relative impedancethrough the medium 180 (i.e., from the inner surface 208 of theelectrode activation substrate 206 to the top electrode 210 of the cover110) at the corresponding DEP electrode region 214. When activated bylight in the light pattern 218, however, the relative impedance throughthe electrode activation substrate 206 is less than the relativeimpedance through the medium 180 at each illuminated DEP electroderegion 214, thereby activating the DEP electrode at the correspondingDEP electrode region 214 as discussed above. DEP electrodes that attractor repel micro-objects (not shown) in the medium 180 can thus beselectively activated and deactivated at many different DEP electroderegions 214 at the inner surface 208 of the electrode activationsubstrate 206 in the region/chamber 202 in a manner determined by thelight pattern 218.

Examples of microfluidic devices having electrode activation substratesthat comprise phototransistors have been described, for example, in U.S.Pat. No. 7,956,339 (Ohta et al.) (see, e.g., device 300 illustrated inFIGS. 21 and 22, and descriptions thereof), the entire contents of whichare incorporated herein by reference. Examples of microfluidic deviceshaving electrode activation substrates that comprise electrodescontrolled by phototransistor switches have been described, for example,in U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, e.g.,devices 200, 400, 500, 600, and 900 illustrated throughout the drawings,and descriptions thereof), the entire contents of which are incorporatedherein by reference.

In some embodiments of a DEP configured microfluidic device, the topelectrode 210 is part of a first wall (or cover 110) of the enclosure102, and the electrode activation substrate 206 and bottom electrode 204are part of a second wall (or support structure 104) of the enclosure102. The region/chamber 202 can be between the first wall and the secondwall. In other embodiments, the electrode 210 is part of the second wall(or support structure 104) and one or both of the electrode activationsubstrate 206 and/or the electrode 210 are part of the first wall (orcover 110). Moreover, the light source 216 can alternatively be used toilluminate the enclosure 102 from below.

With the microfluidic device 200 of FIGS. 1B-1C having a DEPconfiguration, the motive module 162 can select a micro-object (notshown) in the medium 180 in the region/chamber 202 by projecting a lightpattern 218 into the device 200 to activate a first set of one or moreDEP electrodes at DEP electrode regions 214 a of the inner surface 208of the electrode activation substrate 206 in a pattern (e.g., squarepattern 220) that surrounds and captures the micro-object. The motivemodule 162 can then move the captured micro-object by moving the lightpattern 218 relative to the device 200 to activate a second set of oneor more DEP electrodes at DEP electrode regions 214. Alternatively, thedevice 200 can be moved relative to the light pattern 218.

In other embodiments, the microfluidic device 200 can have a DEPconfiguration that does not rely upon light activation of DEP electrodesat the inner surface 208 of the electrode activation substrate 206. Forexample, the electrode activation substrate 206 can comprise selectivelyaddressable and energizable electrodes positioned opposite to a surfaceincluding at least one electrode (e.g., cover 110). Switches (e.g.,transistor switches in a semiconductor substrate) may be selectivelyopened and closed to activate or inactivate DEP electrodes at DEPelectrode regions 214, thereby creating a net DEP force on amicro-object (not shown) in region/chamber 202 in the vicinity of theactivated DEP electrodes. Depending on such characteristics as thefrequency of the power source 212 and the dielectric properties of themedium (not shown) and/or micro-objects in the region/chamber 202, theDEP force can attract or repel a nearby micro-object. By selectivelyactivating and deactivating a set of DEP electrodes (e.g., at a set ofDEP electrodes regions 214 that forms a square pattern 220), one or moremicro-objects in region/chamber 202 can be trapped and moved within theregion/chamber 202. The motive module 162 in FIG. 1A can control suchswitches and thus activate and deactivate individual ones of the DEPelectrodes to select, trap, and move particular micro-objects (notshown) around the region/chamber 202. Microfluidic devices having a DEPconfiguration that includes selectively addressable and energizableelectrodes are known in the art and have been described, for example, inU.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776(Medoro), the entire contents of which are incorporated herein byreference.

As yet another example, the microfluidic device 200 can have anelectrowetting (EW) configuration, which can be in place of the DEPconfiguration or can be located in a portion of the microfluidic device200 that is separate from the portion which has the DEP configuration.The EW configuration can be an opto-electrowetting configuration or anelectrowetting on dielectric (EWOD) configuration, both of which areknown in the art. In some EW configurations, the support structure 104has an electrode activation substrate 206 sandwiched between adielectric layer (not shown) and the bottom electrode 204. Thedielectric layer can comprise a hydrophobic material and/or can becoated with a hydrophobic material, as described below. For microfluidicdevices 200 that have an EW configuration, the inner surface 208 of thesupport structure 104 is the inner surface of the dielectric layer orits hydrophobic coating.

The dielectric layer (not shown) can comprise one or more oxide layers,and can have a thickness of about 50 nm to about 250 nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer maycomprise a layer of oxide, such as a metal oxide (e.g., aluminum oxideor hafnium oxide). In certain embodiments, the dielectric layer cancomprise a dielectric material other than a metal oxide, such as siliconoxide or a nitride. Regardless of the exact composition and thickness,the dielectric layer can have an impedance of about 10 kOhms to about 50kOhms.

In some embodiments, the surface of the dielectric layer that facesinward toward region/chamber 202 is coated with a hydrophobic material.The hydrophobic material can comprise, for example, fluorinated carbonmolecules. Examples of fluorinated carbon molecules includeperfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™).Molecules that make up the hydrophobic material can be covalently bondedto the surface of the dielectric layer. For example, molecules of thehydrophobic material can be covalently bound to the surface of thedielectric layer by means of a linker such as a siloxane group, aphosphonic acid group, or a thiol group. Thus, in some embodiments, thehydrophobic material can comprise alkyl-terminated siloxane,alkyl-termination phosphonic acid, or alkyl-terminated thiol. The alkylgroup can be long-chain hydrocarbons (e.g., having a chain of at least10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively,fluorinated (or perfluorinated) carbon chains can be used in place ofthe alkyl groups. Thus, for example, the hydrophobic material cancomprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminatedphosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments,the hydrophobic coating has a thickness of about 10 nm to about 50 nm.In other embodiments, the hydrophobic coating has a thickness of lessthan 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of a microfluidic device 200 havingan electrowetting configuration is coated with a hydrophobic material(not shown) as well. The hydrophobic material can be the samehydrophobic material used to coat the dielectric layer of the supportstructure 104, and the hydrophobic coating can have a thickness that issubstantially the same as the thickness of the hydrophobic coating onthe dielectric layer of the support structure 104. Moreover, the cover110 can comprise an electrode activation substrate 206 sandwichedbetween a dielectric layer and the top electrode 210, in the manner ofthe support structure 104. The electrode activation substrate 206 andthe dielectric layer of the cover 110 can have the same compositionand/or dimensions as the electrode activation substrate 206 and thedielectric layer of the support structure 104. Thus, the microfluidicdevice 200 can have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 can comprisea photoconductive material, such as described above. Accordingly, incertain embodiments, the electrode activation substrate 206 can compriseor consist of a layer of hydrogenated amorphous silicon (a-Si:H). Thea-Si:H can comprise, for example, about 8% to 40% hydrogen (calculatedas 100*the number of hydrogen atoms/the total number of hydrogen andsilicon atoms). The layer of a-Si:H can have a thickness of about 500 nmto about 2.0 μm. Alternatively, the electrode activation substrate 206can comprise electrodes (e.g., conductive metal electrodes) controlledby phototransistor switches, as described above. Microfluidic deviceshaving an opto-electrowetting configuration are known in the art and/orcan be constructed with electrode activation substrates known in theart. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), the entirecontents of which are incorporated herein by reference, disclosesopto-electrowetting configurations having a photoconductive materialsuch as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short etal.), referenced above, discloses electrode activation substrates havingelectrodes controlled by phototransistor switches.

The microfluidic device 200 thus can have an opto-electrowettingconfiguration, and light patterns 218 can be used to activatephotoconductive EW regions or photoresponsive EW electrodes in theelectrode activation substrate 206. Such activated EW regions or EWelectrodes of the electrode activation substrate 206 can generate anelectrowetting force at the inner surface 208 of the support structure104 (i.e., the inner surface of the overlaying dielectric layer or itshydrophobic coating). By changing the light patterns 218 (or movingmicrofluidic device 200 relative to the light source 216) incident onthe electrode activation substrate 206, droplets (e.g., containing anaqueous medium, solution, or solvent) contacting the inner surface 208of the support structure 104 can be moved through an immiscible fluid(e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic devices 200 can have an EWODconfiguration, and the electrode activation substrate 206 can compriseselectively addressable and energizable electrodes that do not rely uponlight for activation. The electrode activation substrate 206 thus caninclude a pattern of such electrowetting (EW) electrodes. The pattern,for example, can be an array of substantially square EW electrodesarranged in rows and columns, such as shown in FIG. 2B. Alternatively,the pattern can be an array of substantially hexagonal EW electrodesthat form a hexagonal lattice. Regardless of the pattern, the EWelectrodes can be selectively activated (or deactivated) by electricalswitches (e.g., transistor switches in a semiconductor substrate). Byselectively activating and deactivating EW electrodes in the electrodeactivation substrate 206, droplets (not shown) contacting the innersurface 208 of the overlaying dielectric layer or its hydrophobiccoating can be moved within the region/chamber 202. The motive module162 in FIG. 1A can control such switches and thus activate anddeactivate individual EW electrodes to select and move particulardroplets around region/chamber 202. Microfluidic devices having a EWODconfiguration with selectively addressable and energizable electrodesare known in the art and have been described, for example, in U.S. Pat.No. 8,685,344 (Sundarsan et al.), the entire contents of which areincorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, a powersource 212 can be used to provide a potential (e.g., an AC voltagepotential) that powers the electrical circuits of the microfluidicdevice 200. The power source 212 can be the same as, or a component of,the power source 192 referenced in FIG. 1. Power source 212 can beconfigured to provide an AC voltage and/or current to the top electrode210 and the bottom electrode 204. For an AC voltage, the power source212 can provide a frequency range and an average or peak power (e.g.,voltage or current) range sufficient to generate net DEP forces (orelectrowetting forces) strong enough to trap and move individualmicro-objects (not shown) in the region/chamber 202, as discussed above,and/or to change the wetting properties of the inner surface 208 of thesupport structure 104 (i.e., the dielectric layer and/or the hydrophobiccoating on the dielectric layer) in the region/chamber 202, as alsodiscussed above. Such frequency ranges and average or peak power rangesare known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.),U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No.7,612,355), and US Patent Application Publication Nos. US2014/0124370(Short et al.), US2015/0306598 (Khandros et al.), and US2015/0306599(Khandros et al.).

Sequestration Pens.

Non-limiting examples of generic sequestration pens 224, 226, and 228are shown within the microfluidic device 230 depicted in FIGS. 2A-2C.Each sequestration pen 224, 226, and 228 can comprise an isolationstructure 232 defining an isolation region 240 and a connection region236 fluidically connecting the isolation region 240 to a channel 122.The connection region 236 can comprise a proximal opening 234 to thechannel 122 and a distal opening 238 to the isolation region 240. Theconnection region 236 can be configured so that the maximum penetrationdepth of a flow of a fluidic medium (not shown) flowing from the channel122 into the sequestration pen 224, 226, 228 does not extend into theisolation region 240. Thus, due to the connection region 236, amicro-object (not shown) or other material (not shown) disposed in anisolation region 240 of a sequestration pen 224, 226, 228 can thus beisolated from, and not substantially affected by, a flow of medium 180in the channel 122.

The sequestration pens 224, 226, and 228 of FIGS. 2A-2C each have asingle opening which opens directly to the channel 122. The opening ofthe sequestration pen opens laterally from the channel 122. Theelectrode activation substrate 206 underlays both the channel 122 andthe sequestration pens 224, 226, and 228. The upper surface of theelectrode activation substrate 206 within the enclosure of asequestration pen, forming the floor of the sequestration pen, isdisposed at the same level or substantially the same level of the uppersurface the of electrode activation substrate 206 within the channel 122(or flow region if a channel is not present), forming the floor of theflow channel (or flow region, respectively) of the microfluidic device.The electrode activation substrate 206 may be featureless or may have anirregular or patterned surface that varies from its highest elevation toits lowest depression by less than about 3 microns, 2.5 microns, 2microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns,0.2 microns, 0.1 microns or less. The variation of elevation in theupper surface of the substrate across both the channel 122 (or flowregion) and sequestration pens may be less than about 3%, 2%, 1%. 0.9%,0.8%, 0.5%, 0.3% or 0.1% of the height of the walls of the sequestrationpen or walls of the microfluidic device. While described in detail forthe microfluidic device 200, this also applies to any of themicrofluidic devices 100, 230, 250, 280, 290, 320, 400, 450, 500, 700described herein.

The channel 122 can thus be an example of a swept region, and theisolation regions 240 of the sequestration pens 224, 226, 228 can beexamples of unswept regions. As noted, the channel 122 and sequestrationpens 224, 226, 228 can be configured to contain one or more fluidicmedia 180. In the example shown in FIGS. 2A-2B, the ports 222 areconnected to the channel 122 and allow a fluidic medium 180 to beintroduced into or removed from the microfluidic device 230. Prior tointroduction of the fluidic medium 180, the microfluidic device may beprimed with a gas such as carbon dioxide gas. Once the microfluidicdevice 230 contains the fluidic medium 180, the flow 242 of fluidicmedium 180 in the channel 122 can be selectively generated and stopped.For example, as shown, the ports 222 can be disposed at differentlocations (e.g., opposite ends) of the channel 122, and a flow 242 ofmedium can be created from one port 222 functioning as an inlet toanother port 222 functioning as an outlet.

FIG. 2C illustrates a detailed view of an example of a sequestration pen224 according to the present invention. Examples of micro-objects 246are also shown.

As is known, a flow 242 of fluidic medium 180 in a microfluidic channel122 past a proximal opening 234 of sequestration pen 224 can cause asecondary flow 244 of the medium 180 into and/or out of thesequestration pen 224. To isolate micro-objects 246 in the isolationregion 240 of a sequestration pen 224 from the secondary flow 244, thelength L_(con) of the connection region 236 of the sequestration pen 224(i.e., from the proximal opening 234 to the distal opening 238) shouldbe greater than the penetration depth D_(p) of the secondary flow 244into the connection region 236. The penetration depth D_(p) of thesecondary flow 244 depends upon the velocity of the fluidic medium 180flowing in the channel 122 and various parameters relating to theconfiguration of the channel 122 and the proximal opening 234 of theconnection region 236 to the channel 122. For a given microfluidicdevice, the configurations of the channel 122 and the opening 234 willbe fixed, whereas the rate of flow 242 of fluidic medium 180 in thechannel 122 will be variable. Accordingly, for each sequestration pen224, a maximal velocity V_(max) for the flow 242 of fluidic medium 180in channel 122 can be identified that ensures that the penetration depthD_(p) of the secondary flow 244 does not exceed the length L_(con) ofthe connection region 236. As long as the rate of the flow 242 offluidic medium 180 in the channel 122 does not exceed the maximumvelocity V_(max), the resulting secondary flow 244 can be limited to thechannel 122 and the connection region 236 and kept out of the isolationregion 240. The flow 242 of medium 180 in the channel 122 will thus notdraw micro-objects 246 out of the isolation region 240. Rather,micro-objects 246 located in the isolation region 240 will stay in theisolation region 240 regardless of the flow 242 of fluidic medium 180 inthe channel 122.

Moreover, as long as the rate of flow 242 of medium 180 in the channel122 does not exceed V_(max), the flow 242 of fluidic medium 180 in thechannel 122 will not move miscellaneous particles (e.g., microparticlesand/or nanoparticles) from the channel 122 into the isolation region 240of a sequestration pen 224. Having the length L_(con) of the connectionregion 236 be greater than the maximum penetration depth D_(p) of thesecondary flow 244 can thus prevent contamination of one sequestrationpen 224 with miscellaneous particles from the channel 122 or anothersequestration pen (e.g., sequestration pens 226, 228 in FIG. 2D).

Because the channel 122 and the connection regions 236 of thesequestration pens 224, 226, 228 can be affected by the flow 242 ofmedium 180 in the channel 122, the channel 122 and connection regions236 can be deemed swept (or flow) regions of the microfluidic device230. The isolation regions 240 of the sequestration pens 224, 226, 228,on the other hand, can be deemed unswept (or non-flow) regions. Forexample, components (not shown) in a first fluidic medium 180 in thechannel 122 can mix with a second fluidic medium 248 in the isolationregion 240 substantially only by diffusion of components of the firstmedium 180 from the channel 122 through the connection region 236 andinto the second fluidic medium 248 in the isolation region 240.Similarly, components (not shown) of the second medium 248 in theisolation region 240 can mix with the first medium 180 in the channel122 substantially only by diffusion of components of the second medium248 from the isolation region 240 through the connection region 236 andinto the first medium 180 in the channel 122. In some embodiments, theextent of fluidic medium exchange between the isolation region of asequestration pen and the flow region by diffusion is greater than about90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% offluidic exchange. The first medium 180 can be the same medium or adifferent medium than the second medium 248. Moreover, the first medium180 and the second medium 248 can start out being the same, then becomedifferent (e.g., through conditioning of the second medium 248 by one ormore cells in the isolation region 240, or by changing the medium 180flowing through the channel 122).

The maximum penetration depth D_(p) of the secondary flow 244 caused bythe flow 242 of fluidic medium 180 in the channel 122 can depend on anumber of parameters, as mentioned above. Examples of such parametersinclude: the shape of the channel 122 (e.g., the channel can directmedium into the connection region 236, divert medium away from theconnection region 236, or direct medium in a direction substantiallyperpendicular to the proximal opening 234 of the connection region 236to the channel 122); a width W_(ch) (or cross-sectional area) of thechannel 122 at the proximal opening 234; and a width W_(con) (orcross-sectional area) of the connection region 236 at the proximalopening 234; the velocity V of the flow 242 of fluidic medium 180 in thechannel 122; the viscosity of the first medium 180 and/or the secondmedium 248, or the like.

In some embodiments, the dimensions of the channel 122 and sequestrationpens 224, 226, 228 can be oriented as follows with respect to the vectorof the flow 242 of fluidic medium 180 in the channel 122: the channelwidth W_(ch) (or cross-sectional area of the channel 122) can besubstantially perpendicular to the flow 242 of medium 180; the widthW_(con) (or cross-sectional area) of the connection region 236 atopening 234 can be substantially parallel to the flow 242 of medium 180in the channel 122; and/or the length L_(con) of the connection regioncan be substantially perpendicular to the flow 242 of medium 180 in thechannel 122. The foregoing are examples only, and the relative positionof the channel 122 and sequestration pens 224, 226, 228 can be in otherorientations with respect to each other.

As illustrated in FIG. 2C, the width W_(con) of the connection region236 can be uniform from the proximal opening 234 to the distal opening238. The width W_(con) of the connection region 236 at the distalopening 238 can thus be in any of the ranges identified herein for thewidth W_(con) of the connection region 236 at the proximal opening 234.Alternatively, the width W_(con) of the connection region 236 at thedistal opening 238 can be larger than the width W_(con) of theconnection region 236 at the proximal opening 234.

As illustrated in FIG. 2C, the width of the isolation region 240 at thedistal opening 238 can be substantially the same as the width W_(con) ofthe connection region 236 at the proximal opening 234. The width of theisolation region 240 at the distal opening 238 can thus be in any of theranges identified herein for the width W_(con) of the connection region236 at the proximal opening 234. Alternatively, the width of theisolation region 240 at the distal opening 238 can be larger or smallerthan the width W_(con) of the connection region 236 at the proximalopening 234. Moreover, the distal opening 238 may be smaller than theproximal opening 234 and the width W_(con) of the connection region 236may be narrowed between the proximal opening 234 and distal opening 238.For example, the connection region 236 may be narrowed between theproximal opening and the distal opening, using a variety of differentgeometries (e.g. chamfering the connection region, beveling theconnection region). Further, any part or subpart of the connectionregion 236 may be narrowed (e.g. a portion of the connection regionadjacent to the proximal opening 234).

FIGS. 2D-2F depict another exemplary embodiment of a microfluidic device250 containing a microfluidic circuit 262 and flow channels 264, whichare variations of the respective microfluidic device 100, circuit 132and channel 134 of FIG. 1. The microfluidic device 250 also has aplurality of sequestration pens 266 that are additional variations ofthe above-described sequestration pens 124, 126, 128, 130, 224, 226 or228. In particular, it should be appreciated that the sequestration pens266 of device 250 shown in FIGS. 2D-2F can replace any of theabove-described sequestration pens 124, 126, 128, 130, 224, 226 or 228in devices 100, 200, 230, 280, 290, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200. Likewise, the microfluidic device 250 is another variant of themicrofluidic device 100, and may also have the same or a different DEPconfiguration as the above-described microfluidic device 100, 200 ormicrofluidic devices 230, 280, 290, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200 as well as any of the other microfluidic system componentsdescribed herein.

The microfluidic device 250 of FIGS. 2D-2F comprises a support structure(not visible in FIGS. 2D-2F, but can be the same or generally similar tothe support structure 104 of device 100 depicted in FIG. 1A), amicrofluidic circuit structure 256, and a cover (not visible in FIGS.2D-2F, but can be the same or generally similar to the cover 122 ofdevice 100 depicted in FIG. 1A). The microfluidic circuit structure 256includes a frame 252 and microfluidic circuit material 260, which can bethe same as or generally similar to the frame 114 and microfluidiccircuit material 116 of device 100 shown in FIG. 1A. As shown in FIG.2D, the microfluidic circuit 262 defined by the microfluidic circuitmaterial 260 can comprise multiple channels 264 (two are shown but therecan be more) to which multiple sequestration pens 266 are fluidicallyconnected.

Each sequestration pen 266 can comprise an isolation structure 272, anisolation region 270 within the isolation structure 272, and aconnection region 268. From a proximal opening 274 at the channel 264 toa distal opening 276 at the isolation structure 272, the connectionregion 268 fluidically connects the channel 264 to the isolation region270. Generally, in accordance with the above discussion of FIGS. 2B and2C, a flow 278 of a first fluidic medium 254 in a channel 264 can createsecondary flows 282 of the first medium 254 from the channel 264 intoand/or out of the respective connection regions 268 of the sequestrationpens 266.

As illustrated in FIG. 2E, the connection region 268 of eachsequestration pen 266 generally includes the area extending between theproximal opening 274 to a channel 264 and the distal opening 276 to anisolation structure 272. The length L_(con) of the connection region 268can be greater than the maximum penetration depth D_(p) of secondaryflow 282, in which case the secondary flow 282 will extend into theconnection region 268 without being redirected toward the isolationregion 270 (as shown in FIG. 2D). Alternatively, at illustrated in FIG.2F, the connection region 268 can have a length L_(con) that is lessthan the maximum penetration depth D_(p), in which case the secondaryflow 282 will extend through the connection region 268 and be redirectedtoward the isolation region 270. In this latter situation, the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than themaximum penetration depth D_(p), so that secondary flow 282 will notextend into isolation region 270. Whether length L_(con) of connectionregion 268 is greater than the penetration depth D_(p), or the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than thepenetration depth D_(p), a flow 278 of a first medium 254 in channel 264that does not exceed a maximum velocity V_(max) will produce a secondaryflow having a penetration depth D_(p), and micro-objects (not shown butcan be the same or generally similar to the micro-objects 246 shown inFIG. 2C) in the isolation region 270 of a sequestration pen 266 will notbe drawn out of the isolation region 270 by a flow 278 of first medium254 in channel 264. Nor will the flow 278 in channel 264 drawmiscellaneous materials (not shown) from channel 264 into the isolationregion 270 of a sequestration pen 266. As such, diffusion is the onlymechanism by which components in a first medium 254 in the channel 264can move from the channel 264 into a second medium 258 in an isolationregion 270 of a sequestration pen 266. Likewise, diffusion is the onlymechanism by which components in a second medium 258 in an isolationregion 270 of a sequestration pen 266 can move from the isolation region270 to a first medium 254 in the channel 264. The first medium 254 canbe the same medium as the second medium 258, or the first medium 254 canbe a different medium than the second medium 258. Alternatively, thefirst medium 254 and the second medium 258 can start out being the same,then become different, e.g., through conditioning of the second mediumby one or more cells in the isolation region 270, or by changing themedium flowing through the channel 264.

As illustrated in FIG. 2E, the width W_(ch) of the channels 264 (i.e.,taken transverse to the direction of a fluid medium flow through thechannel indicated by arrows 278 in FIG. 2D) in the channel 264 can besubstantially perpendicular to a width W_(con1) of the proximal opening274 and thus substantially parallel to a width W_(con2) of the distalopening 276. The width W_(con1) of the proximal opening 274 and thewidth W_(con2) of the distal opening 276, however, need not besubstantially perpendicular to each other. For example, an angle betweenan axis (not shown) on which the width W_(con1) of the proximal opening274 is oriented and another axis on which the width W_(con2) of thedistal opening 276 is oriented can be other than perpendicular and thusother than 90°. Examples of alternatively oriented angles include anglesin any of the following ranges: from about 30° to about 90°, from about45° to about 90°, from about 60° to about 90°, or the like.

In various embodiments of sequestration pens (e.g. 124, 126, 128, 130,224, 226, 228, or 266), the isolation region (e.g. 240 or 270) isconfigured to contain a plurality of micro-objects. In otherembodiments, the isolation region can be configured to contain only one,two, three, four, five, or a similar relatively small number ofmicro-objects. Accordingly, the volume of an isolation region can be,for example, at least 1×10⁶, 2×10⁶, 4×10⁶, 6×10⁶ cubic microns, or more.

In various embodiments of sequestration pens, the width W_(ch) of thechannel (e.g., 122) at a proximal opening (e.g. 234) can be within anyof the following ranges: about 50-1000 microns, 50-500 microns, 50-400microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns,50-100 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250microns, 70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns,90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250microns, 100-200 microns, 100-150 microns, and 100-120 microns. In someother embodiments, the width W_(ch) of the channel (e.g., 122) at aproximal opening (e.g. 234) can be in a range of about 200-800 microns,200-700 microns, or 200-600 microns. The foregoing are examples only,and the width W_(ch) of the channel 122 can be in other ranges (e.g., arange defined by any of the endpoints listed above). Moreover, theW_(ch) of the channel 122 can be selected to be in any of these rangesin regions of the channel other than at a proximal opening of asequestration pen.

In some embodiments, a sequestration pen has a height of about 30 toabout 200 microns, or about 50 to about 150 microns. In someembodiments, the sequestration pen has a cross-sectional area of about1×10⁴-3×10⁶ square microns, 2×10⁴-2×10⁶ square microns, 4×10⁴-1×10⁶square microns, 2×10⁴-5×10⁵ square microns, 2×10⁴-1×10⁵ square micronsor about 2×10⁵-2×10⁶ square microns.

In various embodiments of sequestration pens, the height H_(ch) of thechannel (e.g., 122) at a proximal opening (e.g., 234) can be within anyof the following ranges: 20-100 microns, 20-90 microns, 20-80 microns,20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns,40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60microns, or 40-50 microns. The foregoing are examples only, and theheight H_(ch) of the channel (e.g., 122) can be in other ranges (e.g., arange defined by any of the endpoints listed above). The height H_(ch)of the channel 122 can be selected to be in any of these ranges inregions of the channel other than at a proximal opening of ansequestration pen.

In various embodiments of sequestration pens a cross-sectional area ofthe channel (e.g., 122) at a proximal opening (e.g., 234) can be withinany of the following ranges: 500-50,000 square microns, 500-40,000square microns, 500-30,000 square microns, 500-25,000 square microns,500-20,000 square microns, 500-15,000 square microns, 500-10,000 squaremicrons, 500-7,500 square microns, 500-5,000 square microns,1,000-25,000 square microns, 1,000-20,000 square microns, 1,000-15,000square microns, 1,000-10,000 square microns, 1,000-7,500 square microns,1,000-5,000 square microns, 2,000-20,000 square microns, 2,000-15,000square microns, 2,000-10,000 square microns, 2,000-7,500 square microns,2,000-6,000 square microns, 3,000-20,000 square microns, 3,000-15,000square microns, 3,000-10,000 square microns, 3,000-7,500 square microns,or 3,000 to 6,000 square microns. The foregoing are examples only, andthe cross-sectional area of the channel (e.g., 122) at a proximalopening (e.g., 234) can be in other ranges (e.g., a range defined by anyof the endpoints listed above).

In various embodiments of sequestration pens, the length L_(con) of theconnection region (e.g., 236) can be in any of the following ranges:about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns,20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200microns, or about 100-150 microns. The foregoing are examples only, andlength L_(con) of a connection region (e.g., 236) can be in a differentrange than the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can bein any of the following ranges: 20-500 microns, 20-400 microns, 20-300microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns,20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns,40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns,50-80 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80microns, 70-150 microns, 70-100 microns, and 80-100 microns. Theforegoing are examples only, and the width W_(con) of a connectionregion (e.g., 236) at a proximal opening (e.g., 234) can be differentthan the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens, the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can beat least as large as the largest dimension of a micro-object (e.g.,biological cell which may be a T cell, B cell, or an ovum or embryo)that the sequestration pen is intended for. For example, the widthW_(con) a connection region 236 at a proximal opening 234 of ansequestration pen that an oocyte, ovum, or embryo will be placed intocan be in any of the following ranges: about 100 microns, about 110microns, about 120 microns, about 130 microns, about 140 microns, about150 microns, about 160 microns, about 170 microns, about 180 microns,about 190 microns, about 200 microns, about 225 microns, about 250microns, about 300 microns or about 100-400 microns, about 120-350microns, about 140-200-200 300 microns, or about 140-200 microns. Theforegoing are examples only, and the width W_(con) of a connectionregion (e.g., 236) at a proximal opening (e.g., 234) can be differentthan the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens, the width W_(pr) of aproximal opening of a connection region may be at least as large as thelargest dimension of a micro-object (e.g., a biological micro-objectsuch as a cell) that the sequestration pen is intended for. For example,the width W_(pr) may be about 50 microns, about 60 microns, about 100microns, about 200 microns, about 300 microns or may be in a range ofabout 50-300 microns, about 50-200 microns, about 50-100 microns, about75-150 microns, about 75-100 microns, or about 200-300 microns

In various embodiments of sequestration pens, a ratio of the lengthL_(con) of a connection region (e.g., 236) to a width W_(con) of theconnection region (e.g., 236) at the proximal opening 234 can be greaterthan or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. Theforegoing are examples only, and the ratio of the length L_(con) of aconnection region 236 to a width W_(con) of the connection region 236 atthe proximal opening 234 can be different than the foregoing examples.

In various embodiments of microfluidic devices 100, 200, 230, 250, 280,290, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, V_(max) can be setaround 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,or 1.5 microliters/sec.

In various embodiments of microfluidic devices having sequestrationpens, the volume of an isolation region (e.g., 240) of a sequestrationpen can be, for example, at least 5×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶,6×10⁶, 8×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, or 8×10⁸ cubic microns, ormore. In various embodiments of microfluidic devices havingsequestration pens, the volume of a sequestration pen may be about5×10⁵, 6×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 8×10⁶, 1×10⁷, 3×10⁷, 5×10⁷, orabout 8×10⁷ cubic microns, or more. In some other embodiments, thevolume of a sequestration pen may be about 1 nanoliter to about 50nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2nanoliters to about 10 nanoliters.

In various embodiment, the microfluidic device has sequestration pensconfigured as in any of the embodiments discussed herein where themicrofluidic device has about 5 to about 10 sequestration pens, about 10to about 50 sequestration pens, about 100 to about 500 sequestrationpens; about 200 to about 1000 sequestration pens, about 500 to about1500 sequestration pens, about 1000 to about 2000 sequestration pens, orabout 1000 to about 3500 sequestration pens. The sequestration pens neednot all be the same size and may include a variety of configurations(e.g., different widths, different features within the sequestrationpen.

FIG. 2G illustrates a microfluidic device 280 according to oneembodiment. The microfluidic device 280 is illustrated in FIG. 2G is astylized diagram of a microfluidic device 100. In practice themicrofluidic device 280 and its constituent circuit elements (e.g.channels 122 and sequestration pens 128) would have the dimensionsdiscussed herein. The microfluidic circuit 120 illustrated in FIG. 2Ghas two ports 107, four distinct channels 122 and four distinct flowpaths 106. The microfluidic device 280 further comprises a plurality ofsequestration pens opening off of each channel 122. In the microfluidicdevice illustrated in FIG. 2G, the sequestration pens have a geometrysimilar to the pens illustrated in FIG. 2C and thus, have bothconnection regions and isolation regions. Accordingly, the microfluidiccircuit 120 includes both swept regions (e.g. channels 122 and portionsof the connection regions 236 within the maximum penetration depth D_(p)of the secondary flow 244) and non-swept regions (e.g. isolation regions240 and portions of the connection regions 236 not within the maximumpenetration depth D_(p) of the secondary flow 244).

FIGS. 3A through 3B shows various embodiments of system 150 which can beused to operate and observe microfluidic devices (e.g. 100, 200, 230,250, 280, 290, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200) according to thepresent invention. As illustrated in FIG. 3A, the system 150 can includea structure (“nest”) 300 configured to hold a microfluidic device 100(not shown), or any other microfluidic device described herein. The nest300 can include a socket 302 capable of interfacing with themicrofluidic device 320 (e.g., an optically-actuated electrokineticdevice 100) and providing electrical connections from power source 192to microfluidic device 320. The nest 300 can further include anintegrated electrical signal generation subsystem 304. The electricalsignal generation subsystem 304 can be configured to supply a biasingvoltage to socket 302 such that the biasing voltage is applied across apair of electrodes in the microfluidic device 320 when it is being heldby socket 302. Thus, the electrical signal generation subsystem 304 canbe part of power source 192. The ability to apply a biasing voltage tomicrofluidic device 320 does not mean that a biasing voltage will beapplied at all times when the microfluidic device 320 is held by thesocket 302. Rather, in most cases, the biasing voltage will be appliedintermittently, e.g., only as needed to facilitate the generation ofelectrokinetic forces, such as dielectrophoresis or electro-wetting, inthe microfluidic device 320.

As illustrated in FIG. 3A, the nest 300 can include a printed circuitboard assembly (PCBA) 322. The electrical signal generation subsystem304 can be mounted on and electrically integrated into the PCBA 322. Theexemplary support includes socket 302 mounted on PCBA 322, as well.

Typically, the electrical signal generation subsystem 304 will include awaveform generator (not shown). The electrical signal generationsubsystem 304 can further include an oscilloscope (not shown) and/or awaveform amplification circuit (not shown) configured to amplify awaveform received from the waveform generator. The oscilloscope, ifpresent, can be configured to measure the waveform supplied to themicrofluidic device 320 held by the socket 302. In certain embodiments,the oscilloscope measures the waveform at a location proximal to themicrofluidic device 320 (and distal to the waveform generator), thusensuring greater accuracy in measuring the waveform actually applied tothe device. Data obtained from the oscilloscope measurement can be, forexample, provided as feedback to the waveform generator, and thewaveform generator can be configured to adjust its output based on suchfeedback. An example of a suitable combined waveform generator andoscilloscope is the Red Pitaya™.

In certain embodiments, the nest 300 further comprises a controller 308,such as a microprocessor used to sense and/or control the electricalsignal generation subsystem 304. Examples of suitable microprocessorsinclude the Arduino™ microprocessors, such as the Arduino Nano™. Thecontroller 308 may be used to perform functions and analysis or maycommunicate with an external master controller 154 (shown in FIG. 1A) toperform functions and analysis. In the embodiment illustrated in FIG. 3Athe controller 308 communicates with a master controller 154 through aninterface 310 (e.g., a plug or connector).

In some embodiments, the nest 300 can comprise an electrical signalgeneration subsystem 304 comprising a Red Pitaya™ waveformgenerator/oscilloscope unit (“Red Pitaya unit”) and a waveformamplification circuit that amplifies the waveform generated by the RedPitaya unit and passes the amplified voltage to the microfluidic device100. In some embodiments, the Red Pitaya unit is configured to measurethe amplified voltage at the microfluidic device 320 and then adjust itsown output voltage as needed such that the measured voltage at themicrofluidic device 320 is the desired value. In some embodiments, thewaveform amplification circuit can have a +6.5V to −6.5V power supplygenerated by a pair of DC-DC converters mounted on the PCBA 322,resulting in a signal of up to 13 Vpp at the microfluidic device 100.

As illustrated in FIG. 3A, the support structure 300 can further includea thermal control subsystem 306. The thermal control subsystem 306 canbe configured to regulate the temperature of microfluidic device 320held by the support structure 300. For example, the thermal controlsubsystem 306 can include a Peltier thermoelectric device (not shown)and a cooling unit (not shown). The Peltier thermoelectric device canhave a first surface configured to interface with at least one surfaceof the microfluidic device 320. The cooling unit can be, for example, acooling block (not shown), such as a liquid-cooled aluminum block. Asecond surface of the Peltier thermoelectric device (e.g., a surfaceopposite the first surface) can be configured to interface with asurface of such a cooling block. The cooling block can be connected to afluidic path 314 configured to circulate cooled fluid through thecooling block. In the embodiment illustrated in FIG. 3A, the supportstructure 300 comprises an inlet 316 and an outlet 318 to receive cooledfluid from an external reservoir (not shown), introduce the cooled fluidinto the fluidic path 314 and through the cooling block, and then returnthe cooled fluid to the external reservoir. In some embodiments, thePeltier thermoelectric device, the cooling unit, and/or the fluidic path314 can be mounted on a casing 312 of the support structure 300. In someembodiments, the thermal control subsystem 306 is configured to regulatethe temperature of the Peltier thermoelectric device so as to achieve atarget temperature for the microfluidic device 320. Temperatureregulation of the Peltier thermoelectric device can be achieved, forexample, by a thermoelectric power supply, such as a Pololu™thermoelectric power supply (Pololu Robotics and Electronics Corp.). Thethermal control subsystem 306 can include a feedback circuit, such as atemperature value provided by an analog circuit. Alternatively, thefeedback circuit can be provided by a digital circuit.

In some embodiments, the nest 300 can include a thermal controlsubsystem 306 with a feedback circuit that is an analog voltage dividercircuit (not shown) which includes a resistor (e.g., with resistance 1kOhm+/−0.1%, temperature coefficient +/−0.02 ppm/C0) and a NTCthermistor (e.g., with nominal resistance 1 kOhm+/−0.01%). In someinstances, the thermal control subsystem 306 measures the voltage fromthe feedback circuit and then uses the calculated temperature value asinput to an on-board PID control loop algorithm. Output from the PIDcontrol loop algorithm can drive, for example, both a directional and apulse-width-modulated signal pin on a Pololu™ motor drive (not shown) toactuate the thermoelectric power supply, thereby controlling the Peltierthermoelectric device.

The nest 300 can include a serial port 324 which allows themicroprocessor of the controller 308 to communicate with an externalmaster controller 154 via the interface 310 (not shown). In addition,the microprocessor of the controller 308 can communicate (e.g., via aPlink tool (not shown)) with the electrical signal generation subsystem304 and thermal control subsystem 306. Thus, via the combination of thecontroller 308, the interface 310, and the serial port 324, theelectrical signal generation subsystem 304 and the thermal controlsubsystem 306 can communicate with the external master controller 154.In this manner, the master controller 154 can, among other things,assist the electrical signal generation subsystem 304 by performingscaling calculations for output voltage adjustments. A Graphical UserInterface (GUI) (not shown) provided via a display device 170 coupled tothe external master controller 154, can be configured to plottemperature and waveform data obtained from the thermal controlsubsystem 306 and the electrical signal generation subsystem 304,respectively. Alternatively, or in addition, the GUI can allow forupdates to the controller 308, the thermal control subsystem 306, andthe electrical signal generation subsystem 304.

As discussed above, system 150 can include an imaging device 194. Insome embodiments, the imaging device 194 comprises a light modulatingsubsystem 330 (See FIG. 3B). The light modulating subsystem 330 caninclude a digital mirror device (DMD) or a micro shutter array system(MSA), either of which can be configured to receive light from a lightsource 332 and transmits a subset of the received light into an opticaltrain of microscope 350. Alternatively, the light modulating subsystem330 can include a device that produces its own light (and thus dispenseswith the need for a light source 332), such as an organic light emittingdiode display (OLED), a liquid crystal on silicon (LCOS) device, aferroelectric liquid crystal on silicon device (FLCOS), or atransmissive liquid crystal display (LCD). The light modulatingsubsystem 330 can be, for example, a projector. Thus, the lightmodulating subsystem 330 can be capable of emitting both structured andunstructured light. One example of a suitable light modulating subsystem330 is the Mosaic™ system from Andor Technologies™. In certainembodiments, imaging module 164 and/or motive module 162 of system 150can control the light modulating subsystem 330.

In certain embodiments, the imaging device 194 further comprises amicroscope 350. In such embodiments, the nest 300 and light modulatingsubsystem 330 can be individually configured to be mounted on themicroscope 350. The microscope 350 can be, for example, a standardresearch-grade light microscope or fluorescence microscope. Thus, thenest 300 can be configured to be mounted on the stage 344 of themicroscope 350 and/or the light modulating subsystem 330 can beconfigured to mount on a port of microscope 350. In other embodiments,the nest 300 and the light modulating subsystem 330 described herein canbe integral components of microscope 350.

In certain embodiments, the microscope 350 can further include one ormore detectors 348. In some embodiments, the detector 348 is controlledby the imaging module 164. The detector 348 can include an eye piece, acharge-coupled device (CCD), a camera (e.g., a digital camera), or anycombination thereof. If at least two detectors 348 are present, onedetector can be, for example, a fast-frame-rate camera while the otherdetector can be a high sensitivity camera. Furthermore, the microscope350 can include an optical train configured to receive reflected and/oremitted light from the microfluidic device 320 and focus at least aportion of the reflected and/or emitted light on the one or moredetectors 348. The optical train of the microscope can also includedifferent tube lenses (not shown) for the different detectors, such thatthe final magnification on each detector can be different.

In certain embodiments, imaging device 194 is configured to use at leasttwo light sources. For example, a first light source 332 can be used toproduce structured light (e.g., via the light modulating subsystem 330)and a second light source 334 can be used to provide unstructured light.The first light source 332 can produce structured light foroptically-actuated electrokinesis and/or fluorescent excitation, and thesecond light source 334 can be used to provide bright fieldillumination. In these embodiments, the motive module 164 can be used tocontrol the first light source 332 and the imaging module 164 can beused to control the second light source 334. The optical train of themicroscope 350 can be configured to (1) receive structured light fromthe light modulating subsystem 330 and focus the structured light on atleast a first region in a microfluidic device, such as anoptically-actuated electrokinetic device, when the device is being heldby the nest 300, and (2) receive reflected and/or emitted light from themicrofluidic device and focus at least a portion of such reflectedand/or emitted light onto detector 348. The optical train can be furtherconfigured to receive unstructured light from a second light source andfocus the unstructured light on at least a second region of themicrofluidic device, when the device is held by the nest 300. In certainembodiments, the first and second regions of the microfluidic device canbe overlapping regions. For example, the first region can be a subset ofthe second region.

In FIG. 3B, the first light source 332 is shown supplying light to alight modulating subsystem 330, which provides structured light to theoptical train of the microscope 350 of system 355 (not shown). Thesecond light source 334 is shown providing unstructured light to theoptical train via a beam splitter 336. Structured light from the lightmodulating subsystem 330 and unstructured light from the second lightsource 334 travel from the beam splitter 336 through the optical traintogether to reach a second beam splitter (or dichroic filter 338,depending on the light provided by the light modulating subsystem 330),where the light gets reflected down through the objective 336 to thesample plane 342. Reflected and/or emitted light from the sample plane342 then travels back up through the objective 340, through the beamsplitter and/or dichroic filter 338, and to a dichroic filter 346. Onlya fraction of the light reaching dichroic filter 346 passes through andreaches the detector 348.

In some embodiments, the second light source 334 emits blue light. Withan appropriate dichroic filter 346, blue light reflected from the sampleplane 342 is able to pass through dichroic filter 346 and reach thedetector 348. In contrast, structured light coming from the lightmodulating subsystem 330 gets reflected from the sample plane 342, butdoes not pass through the dichroic filter 346. In this example, thedichroic filter 346 is filtering out visible light having a wavelengthlonger than 495 nm. Such filtering out of the light from the lightmodulating subsystem 330 would only be complete (as shown) if the lightemitted from the light modulating subsystem did not include anywavelengths shorter than 495 nm. In practice, if the light coming fromthe light modulating subsystem 330 includes wavelengths shorter than 495nm (e.g., blue wavelengths), then some of the light from the lightmodulating subsystem would pass through filter 346 to reach the detector348. In such an embodiment, the filter 346 acts to change the balancebetween the amount of light that reaches the detector 348 from the firstlight source 332 and the second light source 334. This can be beneficialif the first light source 332 is significantly stronger than the secondlight source 334. In other embodiments, the second light source 334 canemit red light, and the dichroic filter 346 can filter out visible lightother than red light (e.g., visible light having a wavelength shorterthan 650 nm).

Coating Solutions and Coating Agents.

Without intending to be limited by theory, maintenance of a biologicalmicro-object (e.g., a biological cell) within a microfluidic device(e.g., a DEP-configured and/or EW-configured microfluidic device) may befacilitated (i.e., the biological micro-object exhibits increasedviability, greater expansion and/or greater portability within themicrofluidic device) when at least one or more inner surfaces of themicrofluidic device have been conditioned or coated so as to present alayer of organic and/or hydrophilic molecules that provides the primaryinterface between the microfluidic device and biological micro-object(s)maintained therein. In some embodiments, one or more of the innersurfaces of the microfluidic device (e.g. the inner surface of theelectrode activation substrate of a DEP-configured microfluidic device,the cover of the microfluidic device, and/or the surfaces of the circuitmaterial) may be treated with or modified by a coating solution and/orcoating agent to generate the desired layer of organic and/orhydrophilic molecules.

The coating may be applied before or after introduction of biologicalmicro-object(s), or may be introduced concurrently with the biologicalmicro-object(s). In some embodiments, the biological micro-object(s) maybe imported into the microfluidic device in a fluidic medium thatincludes one or more coating agents. In other embodiments, the innersurface(s) of the microfluidic device (e.g., a DEP-configuredmicrofluidic device) are treated or “primed” with a coating solutioncomprising a coating agent prior to introduction of the biologicalmicro-object(s) into the microfluidic device.

In some embodiments, at least one surface of the microfluidic deviceincludes a coating material that provides a layer of organic and/orhydrophilic molecules suitable for maintenance and/or expansion ofbiological micro-object(s) (e.g. provides a conditioned surface asdescribed below). In some embodiments, substantially all the innersurfaces of the microfluidic device include the coating material. Thecoated inner surface(s) may include the surface of a flow region (e.g.,channel), chamber, or sequestration pen, or a combination thereof. Insome embodiments, each of a plurality of sequestration pens has at leastone inner surface coated with coating materials. In other embodiments,each of a plurality of flow regions or channels has at least one innersurface coated with coating materials. In some embodiments, at least oneinner surface of each of a plurality of sequestration pens and each of aplurality of channels is coated with coating materials.

Coating Agent/Solution.

Any convenient coating agent/coating solution can be used, including butnot limited to: serum or serum factors, bovine serum albumin (BSA),polymers, detergents, enzymes, and any combination thereof.

Polymer-Based Coating Materials.

The at least one inner surface may include a coating material thatcomprises a polymer. The polymer may be covalently or non-covalentlybound (or may be non-specifically adhered) to the at least one surface.The polymer may have a variety of structural motifs, such as found inblock polymers (and copolymers), star polymers (star copolymers), andgraft or comb polymers (graft copolymers), all of which may be suitablefor the methods disclosed herein.

The polymer may include a polymer including alkylene ether moieties. Awide variety of alkylene ether containing polymers may be suitable foruse in the microfluidic devices described herein. One non-limitingexemplary class of alkylene ether containing polymers are amphiphilicnonionic block copolymers which include blocks of polyethylene oxide(PEO) and polypropylene oxide (PPO) subunits in differing ratios andlocations within the polymer chain. Pluronic® polymers (BASF) are blockcopolymers of this type and are known in the art to be suitable for usewhen in contact with living cells. The polymers may range in averagemolecular mass M_(w) from about 2000 Da to about 20 KDa. In someembodiments, the PEO-PPO block copolymer can have ahydrophilic-lipophilic balance (HLB) greater than about 10 (e.g. 12-18).Specific Pluronic® polymers useful for yielding a coated surface includePluronic® L44, L64, P85, and F127 (including F127NF). Another class ofalkylene ether containing polymers is polyethylene glycol (PEGM_(w)<100,000 Da) or alternatively polyethylene oxide (PEO,M_(w)>100,000). In some embodiments, a PEG may have an M_(w) of about1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

In other embodiments, the coating material may include a polymercontaining carboxylic acid moieties. The carboxylic acid subunit may bean alkyl, alkenyl or aromatic moiety containing subunit. Onenon-limiting example is polylactic acid (PLA). In other embodiments, thecoating material may include a polymer containing phosphate moieties,either at a terminus of the polymer backbone or pendant from thebackbone of the polymer. In yet other embodiments, the coating materialmay include a polymer containing sulfonic acid moieties. The sulfonicacid subunit may be an alkyl, alkenyl or aromatic moiety containingsubunit. One non-limiting example is polystyrene sulfonic acid (PSSA) orpolyanethole sulfonic acid. In further embodiments, the coating materialmay include a polymer including amine moieties. The polyamino polymermay include a natural polyamine polymer or a synthetic polyaminepolymer. Examples of natural polyamines include spermine, spermidine,and putrescine.

In other embodiments, the coating material may include a polymercontaining saccharide moieties. In a non-limiting example,polysaccharides such as xanthan gum or dextran may be suitable to form amaterial which may reduce or prevent cell sticking in the microfluidicdevice. For example, a dextran polymer having a size about 3 kDa may beused to provide a coating material for a surface within a microfluidicdevice.

In other embodiments, the coating material may include a polymercontaining nucleotide moieties, i.e. a nucleic acid, which may haveribonucleotide moieties or deoxyribonucleotide moieties, providing apolyelectrolyte surface. The nucleic acid may contain only naturalnucleotide moieties or may contain unnatural nucleotide moieties whichcomprise nucleobase, ribose or phosphate moiety analogs such as7-deazaadenine, pentose, methyl phosphonate or phosphorothioate moietieswithout limitation.

In yet other embodiments, the coating material may include a polymercontaining amino acid moieties. The polymer containing amino acidmoieties may include a natural amino acid containing polymer or anunnatural amino acid containing polymer, either of which may include apeptide, a polypeptide or a protein. In one non-limiting example, theprotein may be bovine serum albumin (BSA) and/or serum (or a combinationof multiple different sera) comprising albumin and/or one or more othersimilar proteins as coating agents. The serum can be from any convenientsource, including but not limited to fetal calf serum, sheep serum, goatserum, horse serum, and the like. In certain embodiments, BSA in acoating solution is present in a range of form about 1 mg/mL to about100 mg/mL, including 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or more or anywhere inbetween. In certain embodiments, serum in a coating solution may bepresent in a range of from about 20% (v/v) to about 50% v/v, including25%, 30%, 35%, 40%, 45%, or more or anywhere in between. In someembodiments, BSA may be present as a coating agent in a coating solutionat 5 mg/mL, whereas in other embodiments, BSA may be present as acoating agent in a coating solution at 70 mg/mL. In certain embodiments,serum is present as a coating agent in a coating solution at 30%. Insome embodiments, an extracellular matrix (ECM) protein may be providedwithin the coating material for optimized cell adhesion to foster cellgrowth. A cell matrix protein, which may be included in a coatingmaterial, can include, but is not limited to, a collagen, an elastin, anRGD-containing peptide (e.g. a fibronectin), or a laminin. In yet otherembodiments, growth factors, cytokines, hormones or other cell signalingspecies may be provided within the coating material of the microfluidicdevice.

In some embodiments, the coating material may include a polymercontaining more than one of alkylene oxide moieties, carboxylic acidmoieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, or amino acid moieties. In otherembodiments, the polymer conditioned surface may include a mixture ofmore than one polymer each having alkylene oxide moieties, carboxylicacid moieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, and/or amino acid moieties, which may beindependently or simultaneously incorporated into the coating material.

Covalently Linked Coating Materials.

In some embodiments, the at least one inner surface includes covalentlylinked molecules that provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) within the microfluidic device, providing a conditionedsurface for such cells.

The covalently linked molecules include a linking group, wherein thelinking group is covalently linked to one or more surfaces of themicrofluidic device, as described below. The linking group is alsocovalently linked to a moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s).

In some embodiments, the covalently linked moiety configured to providea layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) may include alkyl orfluoroalkyl (which includes perfluoroalkyl) moieties; mono- orpolysaccharides (which may include but is not limited to dextran);alcohols (including but not limited to propargyl alcohol); polyalcohols,including but not limited to polyvinyl alcohol; alkylene ethers,including but not limited to polyethylene glycol; polyelectrolytes(including but not limited to polyacrylic acid or polyvinyl phosphonicacid); amino groups (including derivatives thereof, such as, but notlimited to alkylated amines, hydroxyalkylated amino group, guanidinium,and heterocylic groups containing an unaromatized nitrogen ring atom,such as, but not limited to morpholinyl or piperazinyl); carboxylicacids including but not limited to propiolic acid (which may provide acarboxylate anionic surface); phosphonic acids, including but notlimited to ethynyl phosphonic acid (which may provide a phosphonateanionic surface); sulfonate anions; carboxybetaines; sulfobetaines;sulfamic acids; or amino acids.

In various embodiments, the covalently linked moiety configured toprovide a layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) in the microfluidicdevice may include non-polymeric moieties such as an alkyl moiety, asubstituted alkyl moiety, such as a fluoroalkyl moiety (including butnot limited to a perfluoroalkyl moiety), amino acid moiety, alcoholmoiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety,sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety.Alternatively, the covalently linked moiety may include polymericmoieties, which may be any of the moieties described above.

In some embodiments, the covalently linked alkyl moiety may comprisescarbon atoms forming a linear chain (e.g., a linear chain of at least 10carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be anunbranched alkyl moiety. In some embodiments, the alkyl group mayinclude a substituted alkyl group (e.g., some of the carbons in thealkyl group can be fluorinated or perfluorinated). In some embodiments,the alkyl group may include a first segment, which may include aperfluoroalkyl group, joined to a second segment, which may include anon-substituted alkyl group, where the first and second segments may bejoined directly or indirectly (e.g., by means of an ether linkage). Thefirst segment of the alkyl group may be located distal to the linkinggroup, and the second segment of the alkyl group may be located proximalto the linking group.

In other embodiments, the covalently linked moiety may include at leastone amino acid, which may include more than one type of amino acid.Thus, the covalently linked moiety may include a peptide or a protein.In some embodiments, the covalently linked moiety may include an aminoacid which may provide a zwitterionic surface to support cell growth,viability, portability, or any combination thereof.

In other embodiments, the covalently linked moiety may include at leastone alkylene oxide moiety, and may include any alkylene oxide polymer asdescribed above. One useful class of alkylene ether containing polymersis polyethylene glycol (PEG M_(w)<100,000 Da) or alternativelypolyethylene oxide (PEO, M_(w)>100,000). In some embodiments, a PEG mayhave an M_(w) of about 1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

The covalently linked moiety may include one or more saccharides. Thecovalently linked saccharides may be mono-, di-, or polysaccharides. Thecovalently linked saccharides may be modified to introduce a reactivepairing moiety which permits coupling or elaboration for attachment tothe surface. Exemplary reactive pairing moieties may include aldehyde,alkyne or halo moieties. A polysaccharide may be modified in a randomfashion, wherein each of the saccharide monomers may be modified or onlya portion of the saccharide monomers within the polysaccharide aremodified to provide a reactive pairing moiety that may be coupleddirectly or indirectly to a surface. One exemplar may include a dextranpolysaccharide, which may be coupled indirectly to a surface via anunbranched linker.

The covalently linked moiety may include one or more amino groups. Theamino group may be a substituted amine moiety, guanidine moiety,nitrogen-containing heterocyclic moiety or heteroaryl moiety. The aminocontaining moieties may have structures permitting pH modification ofthe environment within the microfluidic device, and optionally, withinthe sequestration pens and/or flow regions (e.g., channels).

The coating material providing a conditioned surface may comprise onlyone kind of covalently linked moiety or may include more than onedifferent kind of covalently linked moiety. For example, the fluoroalkylconditioned surfaces (including perfluoroalkyl) may have a plurality ofcovalently linked moieties which are all the same, e.g., having the samelinking group and covalent attachment to the surface, the same overalllength, and the same number of fluoromethylene units comprising thefluoroalkyl moiety. Alternatively, the coating material may have morethan one kind of covalently linked moiety attached to the surface. Forexample, the coating material may include molecules having covalentlylinked alkyl or fluoroalkyl moieties having a specified number ofmethylene or fluoromethylene units and may further include a further setof molecules having charged moieties covalently attached to an alkyl orfluoroalkyl chain having a greater number of methylene orfluoromethylene units, which may provide capacity to present bulkiermoieties at the coated surface. In this instance, the first set ofmolecules having different, less sterically demanding termini and fewerbackbone atoms can help to functionalize the entire substrate surfaceand thereby prevent undesired adhesion or contact with thesilicon/silicon oxide, hafnium oxide or alumina making up the substrateitself. In another example, the covalently linked moieties may provide azwitterionic surface presenting alternating charges in a random fashionon the surface.

Conditioned Surface Properties.

Aside from the composition of the conditioned surface, other factorssuch as physical thickness of the hydrophobic material can impact DEPforce. Various factors can alter the physical thickness of theconditioned surface, such as the manner in which the conditioned surfaceis formed on the substrate (e.g. vapor deposition, liquid phasedeposition, spin coating, flooding, and electrostatic coating). In someembodiments, the conditioned surface has a thickness in the range ofabout 1 nm to about 10 nm; about 1 nm to about 7 nm; about 1 nm to about5 nm; or any individual value therebetween. In other embodiments, theconditioned surface formed by the covalently linked moieties may have athickness of about 10 nm to about 50 nm. In various embodiments, theconditioned surface prepared as described herein has a thickness of lessthan 10 nm. In some embodiments, the covalently linked moieties of theconditioned surface may form a monolayer when covalently linked to thesurface of the microfluidic device (e.g., a DEP configured substratesurface) and may have a thickness of less than 10 nm (e.g., less than 5nm, or about 1.5 to 3.0 nm). These values are in contrast to that of aCYTOP® (Asahi Glass Co., Ltd. JP) fluoropolymer spin coating, which hasa thickness in the range of about 30 nm. In some embodiments, theconditioned surface does not require a perfectly formed monolayer to besuitably functional for operation within a DEP-configured microfluidicdevice.

In various embodiments, the coating material providing a conditionedsurface of the microfluidic device may provide desirable electricalproperties. Without intending to be limited by theory, one factor thatimpacts robustness of a surface coated with a particular coatingmaterial is intrinsic charge trapping. Different coating materials maytrap electrons, which can lead to breakdown of the coating material.Defects in the coating material may increase charge trapping and lead tofurther breakdown of the coating material. Similarly, different coatingmaterials have different dielectric strengths (i.e. the minimum appliedelectric field that results in dielectric breakdown), which may impactcharge trapping. In certain embodiments, the coating material can havean overall structure (e.g., a densely-packed monolayer structure) thatreduces or limits that amount of charge trapping.

In addition to its electrical properties, the conditioned surface mayalso have properties that are beneficial in use with biologicalmolecules. For example, a conditioned surface that contains fluorinated(or perfluorinated) carbon chains may provide a benefit relative toalkyl-terminated chains in reducing the amount of surface fouling.Surface fouling, as used herein, refers to the amount of indiscriminatematerial deposition on the surface of the microfluidic device, which mayinclude permanent or semi-permanent deposition of biomaterials such asprotein and its degradation products, nucleic acids and respectivedegradation products and the like.

Unitary or Multi-Part Conditioned Surface.

The covalently linked coating material may be formed by reaction of amolecule which already contains the moiety configured to provide a layerof organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) in the microfluidicdevice, as is described below. Alternatively, the covalently linkedcoating material may be formed in a two-part sequence by coupling themoiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) to a surface modifying ligand that itself has beencovalently linked to the surface.

Methods of Preparing a Covalently Linked Coating Material.

In some embodiments, a coating material that is covalently linked to thesurface of a microfluidic device (e.g., including at least one surfaceof the sequestration pens and/or flow regions) has a structure ofFormula 1 or Formula 2. When the coating material is introduced to thesurface in one step, it has a structure of Formula 1, while when thecoating material is introduced in a multiple step process, it has astructure of Formula 2.

The coating material may be linked covalently to oxides of the surfaceof a DEP-configured or EW-configured substrate. The DEP- orEW-configured substrate may comprise silicon, silicon oxide, alumina, orhafnium oxide. Oxides may be present as part of the native chemicalstructure of the substrate or may be introduced as discussed below.

The coating material may be attached to the oxides via a linking group(“LG”), which may be a siloxy or phosphonate ester group formed from thereaction of a siloxane or phosphonic acid group with the oxides. Themoiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device can be any of the moietiesdescribed herein. The linking group LG may be directly or indirectlyconnected to the moiety configured to provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device. When the linking group LG isdirectly connected to the moiety, optional linker (“L”) is not presentand n is 0. When the linking group LG is indirectly connected to themoiety, linker L is present and n is 1. The linker L may have a linearportion where a backbone of the linear portion may include 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemicalbonding limitations as is known in the art. It may be interrupted withany combination of one or more moieties selected from the groupconsisting of ether, amino, carbonyl, amido, or phosphonate groups,arylene, heteroarylene, or heterocyclic groups. In some embodiments, thebackbone of the linker L may include 10 to 20 atoms. In otherembodiments, the backbone of the linker L may include about 5 atoms toabout 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms toabout 50 atoms; or about 10 atoms to about 40 atoms. In someembodiments, the backbone atoms are all carbon atoms.

In some embodiments, the moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s) may be added to the surface of the substratein a multi-step process, and has a structure of Formula 2, as shownabove. The moiety may be any of the moieties described above.

In some embodiments, the coupling group CG represents the resultantgroup from reaction of a reactive moiety R_(x) and a reactive pairingmoiety R_(px) (i.e., a moiety configured to react with the reactivemoiety R_(x)). For example, one typical coupling group CG may include acarboxamidyl group, which is the result of the reaction of an aminogroup with a derivative of a carboxylic acid, such as an activatedester, an acid chloride or the like. Other CG may include a triazolylenegroup, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide,an ether, or alkenyl group, or any other suitable group that may beformed upon reaction of a reactive moiety with its respective reactivepairing moiety. The coupling group CG may be located at the second end(i.e., the end proximal to the moiety configured to provide a layer oforganic and/or hydrophilic molecules suitable for maintenance/expansionof biological micro-object(s) in the microfluidic device) of linker L,which may include any combination of elements as described above. Insome other embodiments, the coupling group CG may interrupt the backboneof the linker L. When the coupling group CG is triazolylene, it may bethe product resulting from a Click coupling reaction and may be furthersubstituted (e.g., a dibenzocylcooctenyl fused triazolylene group).

In some embodiments, the coating material (or surface modifying ligand)is deposited on the inner surfaces of the microfluidic device usingchemical vapor deposition. The vapor deposition process can beoptionally improved, for example, by pre-cleaning the cover 110, themicrofluidic circuit material 116, and/or the substrate (e.g., the innersurface 208 of the electrode activation substrate 206 of aDEP-configured substrate, or a dielectric layer of the support structure104 of an EW-configured substrate), by exposure to a solvent bath,sonication or a combination thereof. Alternatively, or in addition, suchpre-cleaning can include treating the cover 110, the microfluidiccircuit material 116, and/or the substrate in an oxygen plasma cleaner,which can remove various impurities, while at the same time introducingan oxidized surface (e.g. oxides at the surface, which may be covalentlymodified as described herein). Alternatively, liquid-phase treatments,such as a mixture of hydrochloric acid and hydrogen peroxide or amixture of sulfuric acid and hydrogen peroxide (e.g., piranha solution,which may have a ratio of sulfuric acid to hydrogen peroxide in a rangefrom about 3:1 to about 7:1) may be used in place of an oxygen plasmacleaner.

In some embodiments, vapor deposition is used to coat the inner surfacesof the microfluidic device 200 after the microfluidic device 200 hasbeen assembled to form an enclosure 102 defining a microfluidic circuit120. Without intending to be limited by theory, depositing such acoating material on a fully-assembled microfluidic circuit 120 may bebeneficial in preventing delamination caused by a weakened bond betweenthe microfluidic circuit material 116 and the electrode activationsubstrate 206 dielectric layer and/or the cover 110. In embodimentswhere a two-step process is employed the surface modifying ligand may beintroduced via vapor deposition as described above, with subsequentintroduction of the moiety configured provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s). The subsequent reaction may be performed by exposingthe surface modified microfluidic device to a suitable coupling reagentin solution.

FIG. 2H depicts a cross-sectional views of a microfluidic device 290having an exemplary covalently linked coating material providing aconditioned surface. As illustrated, the coating materials 298 (shownschematically) can comprise a monolayer of densely-packed moleculescovalently bound to both the inner surface 294 of the substrate 286 andthe inner surface 292 of the cover 288 of the microfluidic device 290.The coating material 298 can be disposed on substantially all innersurfaces 294, 292 proximal to, and facing inwards towards, the enclosure284 of the microfluidic device 290, including, in some embodiments andas discussed above, the surfaces of microfluidic circuit material (notshown) used to define circuit elements and/or structures within themicrofluidic device 290. In alternate embodiments, the coating material298 can be disposed on only one or some of the inner surfaces of themicrofluidic device 290.

In the embodiment shown in FIG. 2H, the coating material 298 can includea monolayer of organosiloxane molecules, each molecule covalently bondedto the inner surfaces 292, 294 of the microfluidic device 290 via asiloxy linker 296. Any of the above-discussed coating materials 298 canbe used (e.g. an alkyl-terminated, a fluoroalkyl terminated moiety, aPEG-terminated moiety, a dextran terminated moiety, or a terminal moietycontaining positive or negative charges for the organosiloxy moieties),where the terminal moiety is disposed at its enclosure-facing terminus(i.e. the portion of the monolayer of the coating material 298 that isnot bound to the inner surfaces 292, 294 and is proximal to theenclosure 284).

In other embodiments, the coating material 298 used to coat the innersurface(s) 292, 294 of the microfluidic device 290 can include anionic,cationic, or zwitterionic moieties, or any combination thereof. Withoutintending to be limited by theory, by presenting cationic moieties,anionic moieties, and/or zwitterionic moieties at the inner surfaces ofthe enclosure 284 of the microfluidic circuit 120, the coating material298 can form strong hydrogen bonds with water molecules such that theresulting water of hydration acts as a layer (or “shield”) thatseparates the biological micro-objects from interactions withnon-biological molecules (e.g., the silicon and/or silicon oxide of thesubstrate). In addition, in embodiments in which the coating material298 is used in conjunction with coating agents, the anions, cations,and/or zwitterions of the coating material 298 can form ionic bonds withthe charged portions of non-covalent coating agents (e.g. proteins insolution) that are present in a medium 180 (e.g. a coating solution) inthe enclosure 284.

In still other embodiments, the coating material may comprise or bechemically modified to present a hydrophilic coating agent at itsenclosure-facing terminus. In some embodiments, the coating material mayinclude an alkylene ether containing polymer, such as PEG. In someembodiments, the coating material may include a polysaccharide, such asdextran. Like the charged moieties discussed above (e.g., anionic,cationic, and zwitterionic moieties), the hydrophilic coating agent canform strong hydrogen bonds with water molecules such that the resultingwater of hydration acts as a layer (or “shield”) that separates thebiological micro-objects from interactions with non-biological molecules(e.g., the silicon and/or silicon oxide of the substrate).

Further details of appropriate coating treatments and modifications maybe found at U.S. application Ser. No. 15/135,707, filed on Apr. 22,2016, and is incorporated by reference in its entirety.

Additional System Components for Maintenance of Viability of Cellswithin the Sequestration Pens of the Microfluidic Device.

In order to promote growth and/or expansion of cell populations,environmental conditions conducive to maintaining functional cells maybe provided by additional components of the system. For example, suchadditional components can provide nutrients, cell growth signalingspecies, pH modulation, gas exchange, temperature control, and removalof waste products from cells.

In Situ-Generated Isolation Structures.

In many applications of microfluidic cell manipulation, it is useful tohave the ability to alter structures within the microfluidicenvironment, based upon optical feedback of the microfluidic contentssuch micro-objects, cells, beads and the like. It had been difficultwithin the microfluidics field to make changes to a microfluidic deviceto alter valving function, to direct flows of media, to direct cells toselected portions of the microfluidic chip, and to select cells usingreal-time information. In addition, it can be desirable to removestructures as part of a method of processing micro-objects. Whileoptically actuated dielectrophoresis or opto-electrowetting cell andfluid manipulation modes are highly useful for many of these functions,having yet another mode of micro-object and media flow manipulation thatprovides real-time ability to change the microfluidic flow region andpen environment within the microfluidic device, and to select, isolateand direct cells and fluidic flow therein, is desirable.

It has been surprisingly discovered that a wide variety of isolationstructures can be generated in situ within a microfluidic (ornanofluidic) device as described herein. In many embodiments, insitu-generated isolation structures may be fabricated in the presence ofbiological cells without disturbing general viability. These insitu-generated isolation structures may be used for selectivelyisolating one cell from a set of cells within a microfluidic device; forselective and reversible valving of media flows, sample-containingflows, or reagent flows; concentration of cells from a dilute inputsource; assaying cells from a clonal population within the same device;controlled laminar flows; selectively mixed laminar flows; or directedcell line development, amongst other uses. Applicant describesmicrofluidic devices, compositions and methods of use for these classesof devices having in situ-generated isolation structures.

A microfluidic (or nanofluidic) device is provided which includes anenclosure comprising a substrate, a flow region located within theenclosure, and at least one in situ-generated isolation structuredisposed on the substrate. The in situ-generated isolation structure mayinclude a solidified polymer network. The solidified polymer network mayinclude a photoinitiated polymer. In some embodiments, the solidifiedpolymer network does not include a silicone polymer. In someembodiments, the solidified polymer network does not include silicon. Insome embodiments, the solidified polymer network may include athermosenstive polymer. The solidified polymer network may be solidifiedin situ. All or part of the in situ-generated isolation structure mayconsist of the solidified polymer network.

The in situ-generated isolation structure may be a fully enclosedstructure, a structure open at a portion of its periphery large enoughto admit passage of a micro-object, a barrier, or any combinationthereof. In some embodiments, the in situ-generated isolation structurecan be configured like a pen. Some nonlimiting examples are shown inFIGS. 9C, 9D, and 11B. The in situ-generated isolation structure may beconfigured in any convenient shape to isolate one or more micro-objects,or a subset of a plurality of micro-objects. The in situ-generatedisolation structure can be of a size to contain a single cell or maycontain a plurality of cells. An in situ-generated isolation structurecan be configured like a sequestration pen where the sequestration penhas an isolation region and a connection region, and the connectionregion has a proximal opening to the flow region (which can be a flowchannel) and has a distal opening to the isolation region. In someembodiments, the in situ-generated isolation structure may be a penhaving an opening to the flow region/channel but the in situ-generatedpen may not necessarily have the connection region of a sequestrationpen.

An in situ-generated isolation structure may also be an insitu-generated barrier. The in situ-generated pen or barrier may includea plurality of in situ-generated modules which together form the pen orbarrier. In various embodiments, the at least one in situ-generatedisolation structure may include a plurality of in situ-generatedisolation modules disposed in the flow region, where the insitu-generated isolation modules may be configured to substantiallyrestrict passage of micro-objects into, out of, and/or through the atleast one in situ-generated isolation structure in a size dependentmanner. In some embodiments, each of the plurality of in situ-generatedisolation modules may be spaced apart from each other such thatmicro-objects having a diameter of 5 microns or greater may besubstantially prevented from passing into, out of, and/or through the atleast one in situ-generated isolation structure. In some embodiments,the plurality of in situ-generated isolation modules may be configuredto discriminate between two different types of biological micro-objects,allowing a first type of biological micro-object to pass in and out ofthe at least one in situ-generated isolation structure and substantiallypreventing a second type of biological micro-object from passing into,out of, and/or through the at least one in situ-generated isolationstructure. In various embodiments, the plurality of in situ-generatedisolation modules may be configured to substantially prevent passage ofa microbead into, out of, and/or through the at least one insitu-generated isolation structure.

In some embodiments, more than one in situ-generated isolation structuremay be generated in the microfluidic device. The microfluidic device mayhave a plurality of in situ-generated isolation structures. When morethan one in situ-generated isolation structure is generated in amicrofluidic device, there may be more than one kind of insitu-generated isolation structure generated, and in any combination.

The in situ-generated isolation structure may be designed to betemporary or it may be kept in place until the conclusion of theexperiment/assay/sorting/cloning process being performed in themicrofluidic device. The solidified polymer network of the insitu-generated isolation structure may be at least partially removableby application of increased fluid flow through the flow region,hydrolysis, proteolysis, osmotic change, temperature change, or opticalillumination. In some embodiments, at least a portion of the insitu-generated isolation structure may be removable using a flow of afluidic medium in the flow region, for one non-limiting example.

In some embodiments, the microfluidic device may further include aplurality of in situ-generated pens. Each of the plurality of insitu-generated pens may be disposed to be arranged adjacent to eachother. Each of the plurality of in situ-generated pens may have theproximal opening disposed contiguously to each other. In someembodiments, there may be more than one plurality of in situ-generatedpens formed within a flow region or there may be multiple channelshaving in situ-generated pens disposed along each channel. FIGS. 9C, 9D,and 11B show a variety of in situ-generated pens.

The microfluidic (or nanofluidic) device may further include at leastone sequestration pen, which may include an isolation region and aconnection region, where the connection region has a proximal opening tothe flow region and a distal opening to the isolation region. In someembodiments, the sequestration pen may be an in situ-generated isolationstructure. In various embodiments, the at least one sequestration pen isnot an in situ-generated isolation structure. In some embodiments, thein situ-generated isolation structure may include an in situ-generatedbarrier. In some embodiments, the microfluidic device may furtherinclude a plurality of sequestration pens. The device may furtherinclude a microfluidic channel. The plurality of sequestration pens maybe located adjacent to each other along the channel. Each of theplurality of sequestration pens may be aligned in a row, with eachsequestration pen of the plurality opening off of one side of themicrofluidic channel (e.g., opening in a common direction from a walldefining the microfluidic channel). In some embodiments, there may bemore than one plurality of sequestration pens within a flow region orthere may be multiple channels having sequestration pens disposed alongeach channel. When more than one plurality of sequestration pens ispresent within a flow region of a microfluidic device, one or more ofthe pluralities of sequestration pens may be an in situ-generatedisolation structure. Alternatively, within each plurality ofsequestration pens, some or all of the sequestration pens may be insitu-generated sequestration pens.

The proximal opening of the sequestration pen to the flow region may beoriented substantially parallel to a flow of fluidic medium in the flowregion. In some embodiments, the proximal opening of the sequestrationpen to the flow region may be oriented to not directly receive a flow offluidic medium. Fluidic medium in the flow region (or flow channel) mayexchange with the fluidic medium in the isolation region of thesequestration pen substantially only by diffusion. The proximal openingmay be oriented at an angle to the fluidic flow such that a micro-objectis not removed from the sequestration pen, even if it receives someflow. As an in situ-generated isolation structure may be generated inreal time, orientation may not be square to the flow or may be chosen tonot be square to the flow.

In some embodiments, the solidified polymer network may be configured tobe porous to a flow of fluidic medium. The solidified polymer networkmay not be porous to at least a subset of a plurality of micro-objects.In some embodiments, the solidified polymer network is substantiallynon-porous to micro-object having a diameter of greater than about 500nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 micron, 2 microns, 3 microns, 4microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10microns, 11 microns, 12 microns, 13 microns, 14 microns, 15 microns, ormore.

The solidified polymer network may further have at least a portionformed from photoinitiated polymer. In some embodiments, all of thesolidified polymer network may be formed from photoinitiated polymer. Inother embodiments, the solidified polymer network may have at least aportion formed from thermosensitive polymer. In some embodiments, thepolymer of the solidified polymer network may be a synthetic polymer, amodified synthetic polymer, or a biological polymer. The biologicalpolymer may be light or thermally activatable. The synthetic polymermodifications may include size modification motifs, cleavage motifs, orcell recognition motifs. In some embodiments, the polymer may be amodified polyethylene glycol. The solidified polymer network may be anysuitable polymer described herein and more fully discussed below

The microfluidic device may further include a thermal pad. The thermalpad may be disposed on the substrate at a location of the insitu-generated isolation structure. The thermal pad may include amaterial that has a high thermal conductivity, and optionally, absorbsvisible and/or infrared electromagnetic radiation. The thermal pad maybe created by deposing a metal shape onto the substrate. The thermal padcan comprise any type of metal that can be excited by a light source toproduce heat. Suitable metals include chromium, gold, silver, aluminum,indium tin oxide, or any combination thereof. Metals may be combined ina multi-layered thermal pad, e.g., a layer of chromium, a layer oftitanium, a layer of gold. Other metals (and alloys) are known in theart. The thermal pad can comprise a continuous metal surface or cancomprise a pattern of metal (e.g. metal shapes such as dots, squares,lines, cones, irregular forms). In some embodiments, a thermal pad maybe located beneath all or part of a location where an in situ-generatedisolation will be/has been generated. The thermal pad may be used togenerate heat to gel, swell, reduce, or remove an in situ-generatedisolation structure. Heat may be generated by directing light into themicrofluidic device at the location where such gelling, swelling,reduction or removal is desired. Alternatively, the heat may begenerated electrically (e.g., by an electrical resistor that is part ofor coupled to the thermal pad).

The microfluidic device may include a cover, which may be substantiallytransparent to illumination having wavelengths in the range forphotoactivation of the polymer to form the solidified polymer network ofthe in situ-generated isolation structures. The cover may also besubstantially transparent to illumination in the range suitable forphotocleavage and degradation of an in situ-generated isolationstructure, thereby allowing the reduction and/or removal of thestructure. In various embodiments, the cover may transmit more thanabout 40%, 50%, 60%, 70%, 80%, or 90% of the light directed through it.A cover may have a lower percentage of light transmission and still beutilizable by increasing the time of exposure.

The enclosure of the microfluidic (or nanofluidic) device may furtherinclude a selection sector. In some embodiments, the enclosure may alsoinclude an isolation sector. The flow region may be part of theselection sector and may further extend into the isolation sector. Theflow region may be configured as a channel which may be disposed ineither the selection sector, the isolation sector, or both. In someembodiments, the flow region may not have a channel in the selectionsector but may have a channel in the isolation sector. In otherembodiments, the flow region occupies both the selection sector and theisolation sector. An in situ-generated isolation structure may bedisposed in the selection sector. FIGS. 5A, 5B, 6, 7, 8, 9A, 9B, 11B,13A-C, and 17, are some examples of in situ-generated isolationstructure in the selection sector.

In other embodiments, an in situ-generated isolation structure may bedisposed in the isolation sector. FIGS. 9C, 9D, 10C, 12B, 16, and 19Bshow non-limiting examples of an isolation structure located in theisolation sector. The isolation sector may include at least onesequestration pen, which may be further disposed along a channel. Insome embodiments, the isolation section may include a plurality ofsequestration pens. One or more sequestration pens in the isolationsector may be in situ-generated isolation structures. For example,depending on the configuration and dimensions of the in situ-generatedstructures of FIG. 9D or 11B, these structures may be consideredsequestration pens. The isolation sector may include at least onesequestration pen that is not an in situ-generated isolation structure.

An in situ-generated isolation structure may have a plurality of insitu-generated isolation modules disposed in the flow region andconfigured to prevent exit of at least one of a plurality ofmicro-objects. The plurality of in situ-generated isolation modules maybe referred to interchangeably as in situ-generated barrier modules andthe overall in situ-generated isolation structure formed by the insitu-generated isolation modules may be a member of a group of insitu-generated isolation structures referred to herein as insitu-generated barriers. FIGS. 6, 7, 8A, 8B, 9A-C, show non-limitingexamples. In situ-generated isolation modules may be used todifferentially permit passage of smaller micro-objects while retainingthe larger micro-objects (e.g., biological cells). For example,micro-objects such as beads or microbeads may have a diameter in therange of about 1 micron to about 5 microns, about 5 microns to about 10microns, or about 5 microns to about 15 microns. In contrast, biologicalcells may have a diameter, for example, of about 2 microns to about 5microns for bacterial cells, about 9 microns to about 30 microns forEukaryotic animal somatic cells, about 10 microns to about 100 micronsfor Eukaryotic plant cells, and about 100 microns for human oocytes.Each of the plurality of in situ-generated isolation modules may bespaced apart from each other at a distance to prevent micro-objects of acertain diameter from passing into, out of, and/or through the insitu-generated barrier so formed. The size of the openings between thein situ-generated barrier modules may be sized so that at least onesubset of the plurality of micro-objects are prevented from exiting thein situ-generated isolation structure. For one non-limiting example,beads or microbeads having a diameter of less than about 10 microns maypass through the in situ-generated barrier having modules spaced about10 microns apart, while human cells having a diameter of 10 microns ormore may be prevented from passing into, out of, and/or through the insitu-generated barrier modules. Thus, the in situ-generated isolationstructure may be a in situ-generated barrier acting to sort one type ofmicro-object from second type of micro-object where the micro-objectshave a diameter in the range of about 1 microns to about 20 microns. Asample containing multiple types of micro-objects, including, forexample differently sized biological cells, may be sorted by introducingthe sample into the flow region of the microfluidic device into thesector having an in situ-generated barrier comprising barrier modules.The barrier may be sized to permit the smaller cells to pass through thegaps in the barrier, while preventing the larger cells from passingthrough the in situ-generated barrier. In some embodiments, the one typeof micro-object that is permitted to pass through the in situ-generatedbarrier may be a bead rather than a biological cell. The insitu-generated barrier having in situ-generated barrier modules may belocated in the selection sector.

In some embodiments, the enclosure may include a flow region, at leastone sequestration pen having a proximal opening to the microfluidicchannel, and an in situ-generated barrier, where the flow region furtheris or further has a microfluidic channel. The in situ-generated barriermay be at least part of an in situ-generated isolation structure, andmay act to isolate a micro-object within the sequestration pen or toisolate selected sequestration pens from other sequestration pens. FIGS.4, 5A and B, 8A and B, 10A-C, 12A and B, 13A-C, 14A and B, 15, 16, 17,19B, 21A-C, and 22 show non-limiting examples of such configurations.

The in situ-generated barrier may be located within the enclosure, in aselection sector or an isolation sector, if present, to provide at leasta partial blockade of one of the microfluidic channel or thesequestration pen. In some embodiments, the in situ-generated barriermay be located in the isolation region of the sequestration pen. FIGS.10A-C and 12B show non-limiting examples of in situ-generated barriersin an isolation region of a sequestration pen. In some embodiments, awidth of the in situ-generated barrier is about ¼ to about ¾, about ¼ toabout ½, or about ¼ to about ⅝ of a width of the isolation region. Awidth of the in situ-generated barrier across the isolation region maybe about 3 microns to about 50 microns, about 5 microns to about 40microns, about 5 microns to about 30 microns, about 5 microns to about20 microns, or about 7 microns to about 2 microns. A width of theisolation region may be about 30 microns to about 50 microns, about 20microns to about 40 microns, about 30 microns to about 60 microns, about30 microns to about 90 microns, about 30 microns to about 120 microns,or about 30 microns to about 250 microns. A size of the insitu-generated barrier may be reduced by temperature change or opticalillumination sufficiently to permit the isolated micro-object to exitpast the reduced in situ-generated barrier.

The barrier may further include a capture moiety configured to captureat least one subset of micro-objects disposed in a sequestration pen ora sector that the barrier surrounds. FIGS. 10A-C shows one non-limitingexample of a barrier having a capture moiety incorporated therein, whichmay include, but is not limited to an antibody, a peptide/proteinincluding a binding motif, an oligonucleotide, an oligosaccharide, orany combination thereof.

In other embodiments, the in situ-generated barrier may be disposedwithin the connection region of the sequestration pen. FIGS. 4, 16, 21and 22 show non-limiting examples of such in situ-generated barriers.The in situ-generated barrier may have a dimension across a width of theconnection region of the sequestration pen sized to block exit of atleast one subset of a plurality of micro-objects disposed in theisolation region of the sequestration pen. The at least one subset ofmicro-objects may be biological cells, and may further be one type ofbiological cells that are blocked by the in situ-generated barrier. Inother embodiments, the barrier may be sized to permit exit of a bead.The barrier may further include a capture moiety, which may include, butis not limited to an antibody, a peptide/protein including a bindingmotif, an oligonucleotide, an oligosaccharide, or any combinationthereof, configured to capture at least one subset of micro-objectsdisposed in a sequestration pen (or connection region thereof) or asector that the barrier surrounds. FIGS. 10A-C shows one non-limitingexample of a barrier having a capture moiety incorporated therein. Insome embodiments, a portion of the in situ-generated barrier may extendfrom within the connection region into the microfluidic channel. In someembodiments, the portion of the in situ-generated barrier extending intothe microfluidic channel comprises less than about 50%, about 40%, about30%, about 20%, about 10% or about 5% of a volume of the insitu-generated barrier.

In some embodiments, a width of the in situ-generated barrier is about ¼to about ¾, about ¼ to about ½, or about ¼ to about ⅝ of a width of theconnection region. A width of the in situ-generated barrier across theconnection region may be about 3 microns to about 50 microns about 5microns to about 40 microns, about 5 microns to about 30 microns, about5 microns to about 20 microns, or about 7 microns to about 25 microns. Awidth of the connection region may be about 30 microns to about 50microns, about 20 microns to about 40 microns, about 30 microns to about60 microns, or about 30 microns to about 90 microns. In someembodiments, the in situ-generated barrier may be configured have afirst state and a second state, wherein when the in situ-generatedbarrier is in the first state, it is configured to prevent exit of atleast one subset of the plurality of micro-objects from thesequestration pen and when the in situ-generated barrier is in thesecond state it is configured to permit the at least one subset to passout of the sequestration pen. In various embodiments, the insitu-generated barrier is configured have a first state and a secondstate, where when the in situ-generated barrier is in the first state,it has a size configured to prevent exit of at least one subset of theplurality of micro-objects having a diameter of between 1 microns to 20microns from the sequestration pen, and when the in situ-generatedbarrier is in the second state it has a size configured to permit the atleast one subset of the plurality of micro-objects to pass out of thesequestration pen. A size of the in situ-generated barrier may bereduced by temperature change or optical illumination sufficiently topermit the isolated micro-object to exit past the reduced barrier. FIGS.4, 21 and 22 show nonlimiting examples of an in situ-generated barrierin a connection region of a sequestration pen. The size of one of thedimensions of the in situ-generated barrier may be configured to besufficiently reducible to permit exit of the at least one subset of theplurality of micro-objects. A size of the in situ-generated barrier canbe reducible upon application of increased fluid flow through the flowregion, hydrolysis, proteolysis, osmotic change, temperature change, oroptical illumination. FIG. 16 shows one non-limiting example.

In other embodiments, the in situ-generated barrier is disposed in themicrofluidic channel. FIGS. 5A and B, 6, 7, 8A and B, 13A-C, 14A and B,and 15 show non-limiting examples of an in situ-generated barrier thatis located in a channel of a microfluidic device. In some embodiments,the in situ-generated barrier that is located in a channel, may extendinto one or more sequestration pens, such as for example, FIGS. 5A andB, 21A-C, and 22.

In some embodiments, the in situ-generated barrier may be located closeto or at a proximal opening of a sequestration pen. FIGS. 5A and B,21A-C, and 22 show non-limiting examples. The in situ-generated barriermay be disposed at the edge of the proximal opening of a selectedsequestration pen of the plurality of sequestration pens. The edge canbe a distal edge (as determined relative to a direction of intended flowof medium in the flow region/channel). One non-limiting example is FIGS.8A-C, where the in situ-generated barrier 820, having in situ-generatedbarrier modules 822, with gaps 824 between each of the in sit-generatedbarrier modules may be used to retain larger micro-objects 630, whilepermitting smaller micro-objects 632, 634 to pass through the gaps 824,thereby concentrating and/or sorting the desired micro-objects 630. Thepen selected to be the site of solidification of the in situ-generatedbarrier may be the sequestration pen located at the end of the row ofsequestration pens.

In some embodiments, where a plurality of sequestration pens is present,the plurality of sequestration pens may form a row along the channel.The in situ-generated barrier may prevent at least one subset of aplurality of micro-objects having a diameter of between 1 microns to 20microns from moving past the in situ-generated barrier in the channel.FIGS. 5A and B, 8A and B, and 13A-C, 14B and 15 show non-limitingexamples. In some embodiments, the in situ-generated barrier is locatedat a distal edge of the proximal opening of a sequestration pen locatedat the end of the row of sequestration pens.

In some embodiments, the in situ-generated barrier may include aplurality of in situ-generated barrier modules disposed in the channel.The in situ-generated barrier may be porous to a flow of fluidic medium,but still prevent at least one subset of micro-objects from moving pastthe barrier. The in situ-generated barrier may include a plurality of insitu-generated barrier modules disposed in the microfluidic channel,which permits fluidic medium to pass through the gaps between theplurality of in situ-generated barrier modules. Alternatively, thebarrier may extend from one wall of the microfluidic channel (orproximal openings of sequestration pens) to the opposing wall of themicrofluidic channel, while being porous to a fluidic medium. FIG. 7shows one such in situ-generated barrier. The in situ-generated barrier720 may be porous to a fluidic medium but not permit at least one typeof micro-object to pass into, out of and/or through the insitu-generated barrier 720. A porous in situ-generated barrier may belocated within a microfluidic channel 264 having no sequestration pensadjacent to the in situ-generated porous in situ-generated barrier asshown in FIG. 7, where the porous in situ-generated barrier mayconcentrate and/or sort a sample containing a multiplicity ofmicro-objects of differing sized. A porous in situ-generated barrier mayalso be located within a microfluidic channel having one or moresequestration pens opening off one (or both) sides of the microfluidicchannel, where the porous barrier may concentrate and/or sortmicro-objects and may further retain a sub-set of micro-objects forsubsequent placement within sequestration pens just above the locationof the porous in situ-generated barrier along the microfluidic channel.

The in situ-generated barrier may be disposed at one edge of theproximal opening of a selected pen of the plurality of pens. FIGS. 8Aand B shows non-limiting examples. Alternatively, the in situ-generatedbarrier may be disposed at the distal edge of the proximal opening of afirst (or an outermost) sequestration pen of a row of pens. FIGS. 13A-Cshow one non-limiting example in which in situ-generated barriers aredisposed at the distal edge of the proximal opening of both the firstand last sequestration pens in a row of pens.

In some embodiments, there may be a first plurality of pens and a firstchannel, and additionally, at least a second plurality of pens disposedalong a second channel. FIGS. 13A-C show one non-limiting example. Thein situ-generated barrier may be located at a distal edge of the firstsequestration pen of the first plurality of pens in the first channel,and may optionally be non-porous, blocking entry to the entire firstchannel. The barrier may direct all flow to the second (or more)channels within the flow region, thus directing flow and anymicro-objects contained therein to a different portion of the enclosure.This in situ-generated barrier can direct flow away from the firstchannel and may be removed when no longer needed. Either before or afterthe first in situ-generated barrier is removed, a new in situ-generatedbarrier may be introduced in another part of the flow region (e.g., asecond, third, etc. channel), to re-direct flow to another portion ofthe flow region. Many other configurations are possible to use the insitu-generated barriers as mechanisms to direct flow, including sampleflows containing micro-objects, within the flow region or a channel of amicrofluidic device.

In other embodiments, the in situ-generated barrier may block theproximal openings of at least two contiguous sequestration pens. In someembodiments, a portion of the in situ-generated barrier may extend fromthe channel into the connection region. FIGS. 5A and B show onenon-limiting example.

The in situ-generated barrier blocking the proximal openings may have adimension of at least 50 microns to about 500 microns, 50 microns toabout 300 microns, 50 microns to about 200 microns, 70 microns to about500 microns or about 70 microns to about 400 microns. In someembodiments, the barrier may have a dimension of about 50 microns, 70microns, 90 microns, 100 microns, 120 microns, 140 microns, 160 microns,180 microns, 200 microns, 220 microns, 250 microns, 290 microns, 300microns, 320 microns, 340 microns, 360 microns, 380 microns, 400microns, 420 microns, 440 microns, 460 microns, 480 microns, 500microns, or any range defined by two of the foregoing dimensions.

In various embodiments, a microfluidic device is provided, including anenclosure comprising: a substrate; a flow region including amicrofluidic channel configured to contain a fluidic medium; a firstplurality of sequestration pens disposed adjacent to each other suchthat each sequestration pen of the first plurality opens off a firstside of the microfluidic channel; and a second plurality ofsequestration pens disposed adjacent to each other such that eachsequestration pen of the second plurality opens off a second opposingside of the microfluidic channel. One nonlimiting example is shown inFIG. 14A. Each sequestration pen of the first plurality and the secondplurality of sequestration pens can include an isolation region and aconnection region, the connection region having a proximal opening tothe microfluidic channel and a distal opening to the isolation region.The first side of the microfluidic channel may be configured to receivea first fluidic medium, and the second side of the microfluidic channelmay be configured to receive a second fluidic medium. The first fluidicmedium may be introduced into the first side of microfluidic the channelvia a first fluidic inlet and the second fluidic medium is introducedinto the second side of the microfluidic channel via a second fluidicinlet. The first fluidic medium may flow out of the first side of themicrofluidic channel via a first outlet and the second fluidic mediummay flow out of the second side of the microfluidic channel via a secondoutlet; alternatively, the first and second fluidic media may flow outof a single common outlet. The first fluidic medium and the secondfluidic medium may flow in the same direction, along the microfluidicchannel. The proximal opening of each sequestration pen to themicrofluidic channel may be oriented substantially parallel to a flow offluidic medium in the microfluidic channel.

The microfluidic device so configured may be used for culturing andassaying clonal populations of cells, but is not so limited, and may beused for any method of culturing, sorting or assaying. The microfluidicdevice is configured such that a clonal population may be disposed in atleast one of the sequestration pens of the first plurality of pens andone or more cells of the clonal population may be disposed in therespective sequestration pen of the second plurality of sequestrationpens.

The microfluidic device may further include a barrier dividing themicrofluidic channel into a first sub-channel configured to provide afirst sub-flow of fluidic medium past the first plurality ofsequestration pens and a second sub-channel configured to provide asecond sub-flow of fluidic medium past the second plurality ofsequestration pens, wherein the barrier is punctuated by at least onegap aligned between a proximal opening (to the first sub-channel) of thefirst pen of the first plurality of pens and a proximal opening (to thesecond sub-channel) of a first pen of the second plurality of pens. Thebarrier may further include a plurality of gaps along a length of thebarrier in the microfluidic channel. In some embodiments, each gap maybe aligned between a proximal opening (to the first sub-channel) of eachpen of the first plurality of pens and a proximal opening (to the secondsub-channel) of each respective pen of the second plurality of pens.Other arrangements of the plurality of gaps along the length of thebarrier are possible. For example, each of the plurality of gaps alongthe barrier may be offset from the proximal opening (to the firstsub-channel) of each sequestration pen of the first plurality ofsequestration pens and the proximal opening (to the second sub-channel)of the respective sequestration pen of the second plurality ofsequestration pens. The barrier may have a length that extends from afirst end of the channel to a second end of the channel. The barrier maybe a permanent barrier and may be formed from the same microfluidiccircuit materials that form the sequestration pens and/or channel walls.The one or more cells of the clonal population may be moved from thesequestration pen of the parent clonal population to the respective penof the second plurality of sequestration pens by being transportedthrough the gap aligned with the pen of the first plurality and the penof the second plurality. When the barrier is a permanent barrier havingone or more gaps along its length in the channel, polymerization may beactivated at the one or more gaps, to introduce one or more insitu-generated barriers closing the one or more gaps along its length,where the in situ-generated barriers may comprise a solidified polymernetwork like any described herein. The solidification of the one or moregaps may separate the first sub-channel from the second sub-channel, andprevent cells from moving from the first sub-channel to the secondsub-channel, and vice versa. In some embodiments, the microfluidicdevice comprises a plurality of in situ-generated barriers, closing aplurality of gaps in the barrier punctuated by a plurality of gaps.

In other embodiments, the microfluidic device includes an insitu-generated barrier, wherein the in situ-generated barrier isdisposed along a length of the microfluidic channel, dividing themicrofluidic channel into a first sub-channel configured to provide afirst sub-flow of fluidic medium past the first plurality ofsequestration pens and a second sub-channel configured to provide asecond sub-flow of fluidic medium past the second plurality ofsequestration pens. In some embodiments, the in situ-generated barrierprevents cells from moving from the first sub-channel to the secondsub-channel, and vice versa. FIG. 14B shows one non-limiting example. Inother embodiments, the in situ-generated barrier comprises one or moregaps, as discussed above in the context of permanent barriers. FIG. 15shows one non-limiting example.

Processing of the one or more cells (e.g., one or more cells taken froma clonal population) may be performed in a sequestration pen of thesecond plurality of sequestration pens. The processing may be performedwithout disruption of the ongoing culture conditions of the parentclonal population located in the corresponding sequestration pen of thefirst plurality of sequestration pens.

In other embodiments, a microfluidic device is provided, which includesan enclosure having: a substrate; a channel; at least one sequestrationpen; and an in situ-generated barrier. The sequestration pen may includean isolation region and a connection region, the connection regionhaving a proximal opening to the channel and a distal opening to theisolation region. FIGS. 4, 5, 8A and B, 10A-C, 12A and B, 13A-C, 14A andB, 15, 16, 19A and B, 21A-C and 22 show non-limiting examples. The insitu-generated barrier may be located within the enclosure to provide atleast a partial blockade of the channel and/or one or more of the atleast one sequestration pen. The in situ-generated barrier may includean in situ-generated solidified polymer network. The solidified polymernetwork may include a photoinitiated polymer. The solidified polymernetwork may include a temperature sensitive polymer. The device mayfurther include a substrate having a thermal pad disposed on thesubstrate at a location beneath the solidified polymer network. Thethermal pad may be used to assist gelling, swelling, reduction orremoval of the barrier. In one non-limiting example, the device shown inFIGS. 13A-C may include one or more metal pads, such as gold, to assistin forming and removing the in situ-generated barriers shown.

The in situ-generated barrier may be disposed in the isolation region ofthe sequestration pen. The in situ-generated barrier disposed in anisolation region of a sequestration pen may have a size as describedabove. In some embodiments, the size of the in situ-generated barriermay be reduced by temperature change or optical illumination. The insitu-generated barrier may further include a capture moiety configuredto capture at least one micro-object disposed in the sequestration pen.FIG. 10C shows one such exemplary barrier.

The in situ-generated barrier may be disposed within the connectionregion of the sequestration pen. The in situ-generated barrier may havea dimension across a width of the connection region of the sequestrationpen sized to block exit of at least one subset of a plurality ofmicro-objects disposed in the isolation region of the sequestration pen.The in situ-generated barrier may be sized to block exit of a biologicalmicro-object. The in situ-generated barrier may be sized to permit exitof a bead. The in situ-generated barrier may further include a capturemoiety configured to capture at least one micro-object disposed in thesequestration pen. The in situ-generated barrier disposed in aconnection region of a sequestration pen may have a size as describedabove. A portion of the in situ-generated barrier may extend from theconnection region into the channel. The portion of the in situ-generatedbarrier extending into the channel may be less than 50% of a volume ofthe barrier

In some embodiments, the in situ-generated barrier may be configuredhave a first state and a second state, wherein when the insitu-generated barrier is in the first state, it is configured toprevent exit of at least one subset of the plurality of micro-objectsfrom the sequestration pen and when the in situ-generated barrier is inthe second state it is configured to permit the at least one subset topass out of the sequestration pen. In the first state, the insitu-generated barrier may have a larger size to prevent exit of thesubset of micro-objects. In the second state, the size of the insitu-generated barrier may be at least reduced to permit exit of the atleast one subset of the plurality of micro-objects. A size of the insitu-generated barrier may be at least reduced by application ofincreased fluid flow through the flow region, hydrolysis, proteolysis,osmotic change, temperature change, or optical illumination.

In various embodiments, the in situ-generated barrier may be disposed inthe microfluidic channel. The barrier may be located at one edge of theproximal opening of the sequestration pen, and may extend from theproximal opening across the microfluidic channel. The in situ-generatedbarrier may prevent at least one subset of a plurality of micro-objectshaving a diameter of between 1 micron to 20 microns from moving past thebarrier in the microfluidic channel. In various embodiments, the insitu-generated barrier may include a plurality of in situ-generatedbarrier modules disposed in the microfluidic channel. The insitu-generated barrier may be porous to a fluidic medium. The at leastone sequestration pen may further include a plurality of sequestrationpens. The plurality of sequestration pens may form a row along themicrofluidic channel. The in situ-generated barrier may be disposed atthe distal edge of the proximal opening of a selected sequestration penof the plurality of sequestration pens. The pen selected to be the siteof solidification of the in situ-generated barrier may be thesequestration pen located at the end of the row of sequestration pens.The barrier may be disposed at a distal edge of the proximal opening ofa first sequestration pen of the plurality of sequestration pens. Thebarrier may prevent at least one subset of a plurality of micro-objectsfrom moving into, out of, and/or through the barrier in the microfluidicchannel.

The in situ-generated barrier may include a plurality of insitu-generated barrier modules disposed in the microfluidic channel,which permits fluidic medium to pass through the gaps between theplurality of in situ-generated barrier modules.

The plurality of pens may be a first plurality of pens and the channelis a first channel, and the device further comprises a second pluralityof pens disposed along a second channel. The in situ-generated barriermay be located at a distal edge of the proximal opening of the firstsequestration pen of the first plurality of sequestration pens. Whenconfigured in this manner, the barrier may block the entry of anymicro-objects into the first channel, and direct flow of fluidic mediumand any micro-objects contained therein to a different portion of theenclosure. The in situ-generated barrier may also or alternatively beformed proximal to the first pen of the second plurality ofsequestration pens, where it may block entry of any micro-objects to thesecond channel.

The in situ-generated barrier may block the proximal openings of atleast two contiguous sequestration pens. The barrier blocking theproximal openings may have a dimension of at least 50 microns to about500 microns across the proximal openings. FIGS. 5A and B shows onenon-limiting example.

In yet another embodiment, the microfluidic device may include a firstplurality of sequestration pens disposed in a row, wherein eachsequestration pen of the first plurality opens off a first side of themicrofluidic channel; and a second plurality of sequestration pensdisposed in a row, wherein each sequestration pen of the secondplurality opens off a second opposing side of the microfluidic channel,wherein the in situ-generated barrier is disposed along a length of themicrofluidic channel, dividing the microfluidic channel into a firstsub-channel configured to provide a first sub-flow of fluidic mediumpast the first plurality of sequestration pens and a second sub-channelconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of sequestration pens, wherein the barrier preventscells from moving from the first sub-channel to the second sub-channel,and vice versa. The in situ-generated barrier may include a plurality ofin situ-generated barrier modules. The in situ-generated barrier modulesmay be spaced apart from each other such that the openings between twomodules is smaller than the size of a selected micro-object, which mayhave a size of about 1-20 microns. The in situ-generated barrier may beporous to a flow of fluidic medium. The in situ-generated barrier may beporous to a fluidic medium but not permit at least one type ofmicro-object to pass into, out of, and/or through the barrier. FIG. 14Bshows one non-limiting example. The first side of the channel may beconfigured to receive a first fluidic medium, the second side of thechannel may be configured to receive a second fluidic medium, and thefirst fluidic medium and the second fluidic medium may each flow alongthe in situ-generated barrier to respective first and second outputs ofthe device. The in situ-generated barrier may be configured to prevent amicro-object from moving from the first sub-channel to the secondsub-channel. The in situ-generated barrier may be reducible byapplication of increased fluid flow through the flow region, hydrolysis,proteolysis, osmotic change, temperature change, or opticalillumination, which may thereby erode the in situ-generated barrier orportions therein. FIG. 15 shows one non-limiting example. For example,proteolysis may degrade the exterior of the barrier, causing erosion.The barrier may be reducible by reducing a size of the barrier. If thebarrier is composed of barrier modules, the barrier may be reducible byremoving one or more of the modules. The barrier may be removable byapplication of increased fluid flow through the flow region, hydrolysis,proteolysis, osmotic change, temperature change, or opticalillumination. Individual barrier modules may be selectively removed,leaving a less restrictive barrier in place. Reducing or removingportions/modules of the in situ-generated barrier may permit differentcomponents of the fluidic media to exchange or may permit differentsubsets of micro-objects to exchange past the in situ-generated barrier.

In any of the embodiments of the microfluidic device having at least onein situ-generated isolation structure, the substrate may be configuredto generate dielectrophoresis (DEP) forces within the enclosure asdescribed herein for microfluidic devices 100, 200, 230, 250, 28, 290,300 and the like. The DEP forces may be optically actuated. In otherembodiments, the substrate of the microfluidic device may be configuredto comprise an opto-electrowetting surface, as described herein, anddescribed in more detail in International Application No.PCT/US2016/059234, filed on Oct. 27, 2016, and its disclosure isherewith incorporated in its entirety by reference. Theopto-electrowetting surface may be photoresponsive and be opticallyactuated. In some embodiments, the opto-electrowetting surface may bephotoconductive.

In any of the embodiments, at least one inner surface of the enclosureof the microfluidic device may include a conditioned surface. The atleast one inner surface may include a surface of the substrate. In someembodiments, all the internal surfaces of the enclosure of themicrofluidic device may include a conditioned surface. In variousembodiments, the conditioned surface may be a covalently modifiedsurface. In various embodiments, the covalently modified surface may behydrophilic.

A fuller understanding of the invention may be had by referring to someof the embodiments described in the following Figures.

FIG. 4 shows precisely formed barriers located inside each sequestrationpen of microfluidic device 400, which may be located within a shortdistance from the top of the pen, which may be employed in methods ofisolating, sorting, or assaying. In some instances, the barrier may beintroduced within the connection region thereof. In one example of aprocess to introduce a polymer barrier within a microfluidic device, asolution containing 10% w/v PEGDA (5 Kd) and 1% photoinitiator (IRGACURE2959, 200 Da) was flowed into the device. After allowing equilibrationfor less than 10 min, the desired region was illuminated with UV lightat approximately 340 nm (+/−20 nm), having a power of 400 mW/cm², for 1second, to initiate polymerization creating a barrier such as thatshown. Several of the precisely formed barriers are shown within whitecircles (for emphasis). Forming barriers in precise locations may beparticularly useful for gravity export of selected cells in the presenceof a second set of cells that will not exit the pen in the presence ofthe in situ-generated barrier. Another variant of this may also includea small portion of polymer barrier protruding into the channel,permitting removal by increased flow in the channel after export of afirst set of cells. In some embodiments, barriers may be formed onlywithin selected sequestration pens, and not within every pen. This maybe used to isolate only selected ones of micro-objects present in thesequestration pens of the device, by generating the in situ-generatedbarrier only within or proximal to the openings of the selectedsequestration pens. In some embodiments, the isolated pens (e.g., thosehaving an in situ-generated barrier) isolate micro-objects that do nothave a desired characteristic, such as production or secretion of abiological product of the micro-object. The remaining micro-objects inremaining un-isolated sequestration pens may be exported from thesequestration pen and may further be exported from the microfluidicdevice. In other embodiments, micro-objects in the one or moresequestration pens having an in situ-generated barrier isolating themicro-objects, may be the micro-objects having a selectedcharacteristic. In this embodiment, the remaining micro-objects in theremaining, non-selected sequestration pens may be exported out of theun-isolated sequestration pens, and may be further exported out of themicrofluidic device. After such export, the in situ-generated barriersmay be removed, permitting further processing of the previously isolatedmicro-objects.

FIGS. 5A and B show an in situ-generated barrier 520 generated across anumber of contiguous pens, where the barrier is shown within the whitecircle in the microfluidic channel. The same conditions for introducingthe polymerizable polymer were used as for the embodiment shown in FIG.4, but the exposure time for this example was 7.5 seconds, and the powerof the UV light was 100 microW/cm², and is discussed in further detailin Example 1 below, and demonstrated that the polymer in situ-generatedbarrier 520 can be introduced and kept in place for several days, whilestill permitting cell growth within the group of sequestration pensisolated by the in situ-generated barrier, as shown in FIG. 5B, wherethe number of biological cells have increased in each pen. An insitu-generated barrier like that of FIGS. 5A and 5B may be used in anyvariety of method to isolate, sort, or assay selectively chosen groupsof biological cells.

FIG. 6 shows an example of an in situ-generated barrier 620 having insitu-generated barrier modules 622 placed into a channel 264, defined bychannel walls 610, of microfluidic device 600. The solidification ofbarrier modules 622 can be performed to leave gaps 624 between eachbarrier module 622 and its adjacent module 622. The flowable polymer maybe introduced within the microfluidic channel 264, and solidified byilluminating selected points within the microfluidic channel 264 tosolidify the polymer network of the in situ-generated barrier 620. Asample containing micro-objects 630 along with undesired materials 632,and 634 may be introduced into the microfluidic device 600 with flow 278towards the barrier 620. The size of the gap can be selected to permitsome species of micro-objects to pass through the gap(s) 624 whilepreventing larger micro-objects such as cells 630 from passing throughthe barrier 620, thereby isolating cells 630 behind the barrier. Smallermicro-objects may include beads (not shown), smaller micro-objects (notshown), cellular debris 632, or micro-objects 634 such as organelles,insoluble proteins, nucleic acids and the like. The barrier 620 maytherefore be used as a sieving/sorting structure to concentrate samplesloaded onto the microfluidic device 600. Once the sample has beenconcentrated, e.g., desired micro-objects 630 have been collected atbarrier 620, the barrier may be removed or reduced sufficiently topermit micro-objects 630 to be moved by any combination of flow, gravityor electrokinetic forces such as DEP forces to another part of themicrofluidic device for further culturing or processing. Alternatively,the concentrated set of micro-objects 630 may be exported by anysuitable motive means out of the microfluidic device 600. Concentrationof micro-objects does not require that only micro-objects 630 areretained by barrier 620, only that a percentage of the undesired 632,634 materials is reduced relative to the sample introduced intomicrofluidic device 600. Further, in some embodiments, the mixture ofmaterials introduced into microfluidic device 600 may not have undesiredmicro-objects 632, 634, but may simply be very dilute. Barrier 620 mayconcentrate a dilute sample containing for example, rare cells, andpermit isolation or export of a concentrated sample of the desiredmicro-objects.

FIG. 7 shows an example of an in situ-generated barrier 720 stretchingsubstantially across a channel 264, defined by channel wall(s) 710, inmicrofluidic device 700. Barrier 720 performs in a similar manner to theembodiment of FIG. 6, differing in that barrier 720 does not havebarrier modules having gaps, but instead has a defined porosity,permitting some components of a sample flowed in with flow 278 acrossbarrier 720 (e.g., materials 632, 634, defined as above) but notpermitting a selected micro-object 630 (e.g., a biological cell) ofinterest across, thereby concentrating or sorting desired micro-objects630 away from other components of a sample.

For the embodiments of FIGS. 6 and/or 7, after the desired cells 630 areconcentrated at the region adjacent to the in situ-generated barrier,the in situ-generated barrier may be removed. For example, thesolidified polymer network of the in situ-generated barrier may besusceptible to photocleavage, when illuminated with light of awavelength configured to cleave portions of the solidified polymernetwork. After removal of the in situ-generated barrier, theconcentrated cells 630 may be moved selectively using any suitablemotive means, including DEP forces (including optically actuated DEP(OET)), gravity or fluidic flow.

FIGS. 8A-C show another exemplar of an in situ-generated barrier 820that can be used to concentrate dilute samples, and additionally assistin selective disposition of micro-objects 630 into selectedsequestration pens 830 in microfluidic device 800. In situ-generatedbarrier 820 is generated in situ within channel 264, defined by channelwall 810 and sequestration pen wall material 812, similarly as in FIGS.4-7 as described above. The barrier 820 includes in situ-generatedbarrier modules 822 spaced apart from each other by gap(s) 824, whichare selected to permit undesired materials 632, 634 (which may be anyundesired materials within a sample, having a size smaller than the sizeof the gap 822). Dilute sample is flowed in within the microfluidicchannel 264 with flow 278 and cells are concentrated at the insitu-generated barrier 820. The flow can be stopped, then theconcentrated cells 630 can be loaded, for example, by flow, gravity, OETforces, or any other suitable method into the sequestration pens 830near the barrier 820. Flow through the channel can be reinstituted todislodge the barrier module 822, or the barrier modules 822 may beremoved by any of optical illumination, hydrolysis, proteolysis, orthermal change. This process can be repeated with a second, newlygenerated barrier 820′ (not shown) located at a second or more set ofpens differing from the location of the first set of pens, in order toload and concentrate multiple dilute aliquots of cell-bearing samples.The multiple aliquots of cell-bearing samples may all be derived fromthe same source (e.g., same mammal, same cell line, and the like) or mayeach be derived from a different one of a mammal, a cell line and thelike.

FIGS. 9A-D shows a microfluidic device 900 having a fluidic inlet 930and a fluidic outlet 932. Flowable polymer is introduced at inlet 930flowed to points within the substrate. An in situ-generated isolationstructure 920 forming a barrier is provided, by activatingsolidification of a plurality of barrier modules 922, having a gap 924between each barrier module and its neighboring barrier module, byilluminating selected portions of the substrate 208. The spacing may beselected to permit materials such as cellular debris 632, ormicro-objects 634 such as organelles, insoluble proteins, nucleic acidsand the like. A sample may be introduced at inlet 930. As the flow 278continues through the barrier 920, undesired materials 632 and 634,which have a size smaller than the gap between the barrier modules 922,may pass through the isolation structure 920, and may be furtherexported out of the microfluidic device via fluidic outlet 932, as shownin FIG. 9B. In a further embodiment, in situ-generated pens 940 may beintroduced as shown in FIG. 9C. Depending on the dimensions of pen 940,it may be an in situ-generated sequestration pen. The isolationstructure 920 may be removed by any suitable method such as opticalillumination (if the solidified polymer network of the barrier modulesis susceptible to photocleavage). The released micro-objects 630 may beselected and moved to be isolated within in situ-generated pens 940, forfurther processing.

FIG. 10 shows the use of small localized in situ-generated barriers1020, acting as pre-selected traps. The barrier traps may befunctionalized in situ-generated barriers, which can have a capturemoiety 1024 such as antibodies or other cell surface recognition motifssuch as an RGD motif peptide. For simplicity, the capture moiety 1024 isshown as an antibody but the in situ generated barrier traps are not solimited. A sample including desired cells 630 and other, not desiredcells 636, can be introduced in fluidic flow 278 within microfluidicchannel 264, and the subset of cells, for example, cells 636, that canbind to the capture moiety 1024 can be immobilized by its interactionwith the capture moiety 1024 on the localized in situ-generated barriers1020. The in situ-generated barrier traps (1020 plus 1024) can belocated either near the proximal opening to the channel within the penor can be located within a more distal section of the connection regionor even within the isolation region of the pen. The remainder of thecells (e.g., cells 630) which do not have any cell surface motif thatcan bind to the in situ-generated barrier trap (1020+1024) can beexported out of the microfluidic device by increasing the flow rate inthe channel, or may be moved to another region within the microfluidicdevice for further processing. In other embodiments, cells 636 may bethe desired portion of the introduced sample flow, and after isolatingcells 636 as described, undesired cells 630 may be exported out of themicrofluidic channel, and optionally, out of the microfluidic device.

The in situ-generated barrier trap (1020+1024) can be formed bycopolymerizing two polymers, one having, for example, an RGD peptidemotif, or by modifying a precursor pre-polymer to have such motif.Another alternative is to immobilize antibodies within the insitu-generated barrier trap or immobilizing the antibodies after thebarrier trap has been formed. In one example, biotinylated orstreptavidin sites can be introduced either throughout the trap or juston the surface of the in situ-generated barrier 1020, and streptavidinor biotin labeled antibodies may associate with the biotin.Alternatively, modified antibodies may be devised, containing aphotoactivatable functionality, such as benzophenone, which may besubjected to photoinitiated insertion into the surface of the polymerbarrier at the same time, or after formation of the in situ-generatedbarrier.

FIGS. 11A and B show pens made upon demand upon loading cells 630 to aflow region 1114, bounded by wall 1110 of a microfluidic device 1100.Before, contemporaneously, or after cells 630 have been introduced, theflowable polymer is also introduced. In situ-generated isolationstructure 1122 and in situ-generated pens 1120 may be formed byilluminating the desired locations on the substrate 208 surface toinitiate polymerization. The in situ-generated pens 1120 and structure1122 may be oriented such that the typical flow direction 278 may notdisturb the cell 630 from its newly encompassing pen/structure. Testing,sorting and culturing may be performed on the cells 630 isolated in thein situ-generated pen 1120 or structure 1122. However, one commondirection of each of the pens 1120 or structure 1122 created around eachof the cells 630 may be open, thus permitting each of the cells 630 tobe exported changing the flow direction, for example, to flush the cellsout of the in situ-generated pens. Alternatively, each cell 630 may beselected and moved individually by, for example, using DEP forces, whichmay be optically actuated.

FIGS. 12A and B show an in situ-generated barrier 1220 introduced withina sequestration pen 830 of microfluidic device 1200 to subdivide theisolation region. One exemplary use may be to controllably remove asubset of the cell population 636, while ensuring that other subsets 630are retained within the pen. Before, contemporaneously, or after cells630 and 636 have been introduced to the flow region 1214 and are thendisposed within the sequestration pen, flowable polymer is introducedinto the isolation region of the sequestration pen 830. Photo-initiatedpolymerization of in situ-generated barrier is performed by illuminatinga selected portion of the substrate 208 at the desired location. Athermoreversible (which could include light actuated thermallyreversible) or photocleavable polymer may be employed to selectivelyremove sections of the in situ-generated barriers thereby permittingselective cell export.

FIG. 13A-C show another form of an in situ-generated barrier 1320 caninclude valve type structures. In situ-generated barriers 1320 can beused to direct flows including cells in to preselected regions of thechip, while blocking them from flowing into nonselected regions. Theselective introduction and removal of in situ-generated barrier 1320 maybe useful for performing multiplex experiments within the microfluidicdevice 1300. Prior to introduction of any cells, flowable polymer may beintroduced at inlet 930. Photoinitiation of polymerization, at selectedpoints of the substrate surface 208, at the distal edge of the openingof the first pen 830 of the first row of sequestration pens 1312 and atthe edge of the opening of the last pen of the row of sequestration pens830, all of which open to first channel 1302, forms a set of insitu-generated barriers 1320 excluding entry of cells to the firstchannel 1302. Similarly, in situ-generated barriers 1320 are introducedat the entrance and exits of third channel 1306 having a third plurality(arranged in a row) of sequestration pens 1316. As shown in FIG. 13A, aflow of cells 630 may be introduced and are directed to flow into thesecond channel 1304. The cells are prevented from entering first channel1302 and from third channel 1306. Cells 630 are constrained to enterun-blocked channel 1304 to be placed into pens 830 within channel 1304.Once delivery of cells 630 is completed, a second set of insitu-generated barriers 1320 may be created at the ends of the secondchannel 1304. The first set of in situ-generated barriers 1320 blockingchannel 1302 and 1306 may then be removed, in any way described herein.A second fluidic flow containing cells 636 may then be introduced viainlet 930. Cells 636 may be constrained to enter channels 1302 and 1306but may not enter channel 1304, as shown in FIG. 13B. Cells not enteringany channel may be swept with fluidic flow to outlet 932. The order ofremoving the first set of in situ-generated barriers 1320 at channels1302 and 1306 and generating the second set of in situ-generatedbarriers 1320 blocking channel 1304 may be reversed. In othervariations, each channel 1302, 1304, 106 may each in turn be madeaccessible to cells being flowed into microfluidic device 1300.

In another variation, microfluidic device 1300 may include thermal pads(not shown) at the points where one wishes to introduce insitu-generated barriers 1320 at channels 1302, 1304, 1306. Heating thethermal pads with a laser to locally increase the temperature, in thepresence of a temperature sensitive polymer, can form a hydrogel in thearea defined by the thermal pad and laser. As the light is removed, itcools and the hydrogel may dissolve.

FIG. 13C shows microfluidic device 1300 having differentially loadedchannels having different cells in each channel, as selected. Thedifferent cells of each channel may be derived from different samples,e.g., different biopsy samples, different clonal populations or any kindof multiplex sample, and any sort of processing may be performed on thecells 630, 636 specifically disposed in microfluidic device 1300.

FIGS. 14A and B show another use for an in situ-generated barrier, whichmay be used to divide more precisely a laminar flow in microfluidicdevice 1400. A known problem with laminar flow is that the laminarnature of the flow fails with distance, and may require rigorousperformance criteria to function at all. In one non-limiting example ofhow an in-situ generated barrier may provide benefit when using laminarflow-dependent methods. In microfluidic device 1400, as shown in FIG.14A, there may be a substrate 208; a flow region including amicrofluidic channel 122 configured to contain a fluidic medium flow278; a first plurality of sequestration pens (830, 831, 832) disposedadjacent to each other such that each sequestration pen of the firstplurality opens off a first side of the microfluidic channel 122; and asecond plurality of sequestration pens (830′, 831′, 832′) disposedadjacent to each other such that each sequestration pen of the secondplurality opens off a second opposing side of the microfluidic channel122. The first and second plurality of pens and the microfluidic circuitmay be made of microfluidic circuit material 1460. The first side of themicrofluidic channel may be configured to receive a first fluidicmedium, and the second side of the microfluidic channel may beconfigured to receive a second fluidic medium. The first fluidic mediummay be introduced into the first side of microfluidic the channel via afirst fluidic inlet 930 and the second fluidic medium is introduced intothe second side of the microfluidic channel via a second fluidic inlet930′. One or more cells may be introduced to pens 830, 831, 832 of thefirst plurality of sequestration pens. In FIG. 14A, a first clonalpopulation of cells 631 may be disposed in sequestration pen 830; asecond clonal population of cells 633 may be disposed in the secondsequestration pen 831; and a third clonal population of cells 635 may bedisposed in sequestration pen 832. In some embodiments, only one cell631, 633, 635 may be provided to respective pens 830, 82, 833 and eachcell may be cultured to provide each respective clonal population. One(or optionally more) cell of each clonal population may be selected andthen delivered across microfluidic channel 122 to a corresponding pen onthe opposite side of the microfluidic device. For example, cell 631′ maybe selected from clonal population of cells 630 in sequestration pen830, and moved, using any suitable motive means including DEP forces(which may be optically actuated) to corresponding sequestration pen830′ of the second plurality of sequestration pens. This may be repeatedfor each respective one or more cell 633′ of clonal population of cell633 in pen 831, and cell 633′ may be delivered to pen 831′, and so on.Flowable polymer may be introduced to the microfluidic channel 122 atthat point, or at any earlier point. An in situ-generated barrier 1420may be then introduced along the length of the microfluidic channel 122,dividing the microfluidic channel into a first sub-channel bordering thefirst plurality of sequestration pens 830, 831, 832 and a secondsub-channel bordering the second plurality of sequestration pens 830′,831′, 832′, and preventing cells from crossing from the firstsub-channel to the second sub-channel, and vice versa. The insitu-generated barrier 1420 may not be porous, and may permit a secondmedium to be introduced in second flow 278′, which may differ in somerespect from a first medium in first flow 278 in the first sub-channel.The second fluidic medium may be introduced to only the one or morecell(s) in each sequestration pen of the second plurality ofsequestration pens, and not to the remainder of the clonal populations.The second medium may contain assay reagents for evaluation of thecell(s) 631′, 633′, 635′. One or more assays may be performed on the setof cell(s) 631′, 633′, 635′ in the second plurality of sequestrationpens or one or more of cell(s) 631′, 633′, 635′ may be exported. Anysort of further processing may be performed on the cells 631′, 633′,635′ which may identify a selected characteristic of the clonalpopulation from which cell 631′, 633′, 635′ is derived.

In some embodiments, the in situ-generated barrier may be removed, and asecond set of one or more cell(s) derived from each or a selected numberof the clonal populations of the first plurality of sequestration pensmay be introduced to the opposing pens. A second in-situ-generatedbarrier may be introduced and subsequent assaying or other processingmay be performed upon them.

In some embodiments, the microfluidic device may have a barrier presentwhen cells are introduced, wherein the barrier is punctuated with one ormore gaps (not shown). The barrier punctuated with one or more gaps maynot be an in situ-generated barrier, but may be formed from the samemicrofluidic circuit material that forms the sequestration pens and themicrofluidic channel wall. The gap(s) are aligned adjacent to theopening of a first pen (e.g., 830) of the first plurality ofsequestration pens and also aligned with the opening of the respectivefirst pen (e.g., 830′) of the second plurality of sequestration pens.The one or more gaps have a size configured to permit the one or morecell of the first clonal population to be selected and moved from thefirst pen of the first plurality of pens on the first side of themicrofluidic channel to the first pen of the second plurality of pens onthe opposing second side of the microfluidic channel. After delivery ofthe cells 631′, 633′, 635′, the one or more gaps may be closed with oneor more in situ-generated barriers as described above. The cells 631′,633′, 635′ may be further processed in any suitable method as describedherein and may be assayed to identify desirable clonal populations ofcells. The desirable clonal population of cells may be exported forfurther expansion or development, or may be cultured in place in thesequestration pens of microfluidic device 1400. Clonal populationsidentified as not desirable may be exported from the sequestration pensand may further be exported from the microfluidic device 1400.

FIG. 15 shows another variation, microfluidic device 1500, of themicrofluidic device 1400. In some embodiments, the in situ-generatedbarrier1500 may additionally have varying degrees of porosity, effectedby either small ruptures in the barrier 1520 or by increasing porosityof the barrier 1520 itself. The in situ-generated barrier may bedisrupted to have increasing spaced in situ-generated barrier modules1522, 1524, 1526, 1528. In this approach, differing amounts of a firstmedium, introduced at first inlet 930, flowing in the first sub-channelin medium flow 278 and a second fluidic medium, introduced at secondinlet 930′, and flowing in the second sub-channel in medium flow 278′,can permeate the barrier and affect the pairs of cells 631, 631′; 633,633′ 635, 635′ in pens on either side of the barrier via diffusion, andmay affect cell development.

Any of these laminar in situ-generated barriers (FIG. 14B or 15) may beuseful for directed cell line development.

FIG. 16 show an in situ-generated polymer barrier that may be swelled orde-swelled using media or solvent changes. After a cell 630 has beendisposed in sequestration pen 830 made of microfluidic circuit material1660 (which may be the same or different from the microfluidic circuitmaterial forming channel wall 1620), and flowable polymer has beenintroduced into the fluidic channel 264, the in situ-generated barrier1620 may be introduced by, for example, illuminated a selected point onthe substrate 208, to effect polymerization of barrier 1620. In someembodiments, the photoinitiated solidified polymer network may beswelled to state 1620′, while performing other sorting or processingsteps on other pens (not shown) in the microfluidic device 1600,preventing exit of the cell 630. The barrier may also prevent solublemedia from entering the pen 830 during the processing steps, shieldingthe cell 630. The in situ-generated barrier may be de-swellled to state1620 and permit exit of cell 630.

FIG. 17 shows a microfluidic device 1700 having a plurality of insitu-generated barrier modules 1720 to 1732 introduced before or afterintroduction of micro-objects 630 and micro-objects 636. Each section ofthe field of the microfluidic device may be examined and tested for adesired micro-object 630. Individual barrier modules may be removed bysuitable methods as described herein, and the micro-objects may besorted to different portions of microfluidic device 1700.

FIG. 18 shows an example of in situ-generated isolation structures usedfor prototyping of features for a microfluidic device 1800. Elementssuch as flow region wall(s) 1810, isolation structure sub-units 1820 and1822, and prototyped channel units 1864, 1865 may be introduced andrecombined rapidly to test new designs by adding the new design aspectsvia in situ-generated barrier/isolation structures. This can be analternative for expensive experimental masks.

FIGS. 19A and 19B show examples of an in situ-generated enclosure1920 ina sequestration pen of a microfluidic device 1900 to define and isolatea selected cell 636. It can also be used to isolate a cell with reagentsto initiate transfection In some embodiments, the enclosure may beswelled to a swelled state 1920′, as described herein, and mayoptionally swell sufficiently to permeabilize the cell 636″. FIGS. 20Aand 20B show a microfluidic device 2000 utilizing a similar enclosure2020 outside of any sequestration pen, to isolate a cell to identify itslocation and optionally to swell (2020′) and permeabilize cell 636″.

The microfluidic device 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 may have any combinationof features, components or dimensions as described for microfluidicdevices 100, 200, 230, 250, 280, 290, 300 and may be suitably used forany method described. Microfluidic device 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 mayfurther be combined with any feature described for a respective other ofmicrofluidic device 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, in anysuitable combination and used in any suitable method described herein,as one of skill would select.

Polymers for Use in the Solidified Polymer Network of the InSitu-Generated Isolation Structure.

In various embodiments of the solidified polymer network of an isolationstructure, the solidified polymer network may be a synthetic polymer, amodified synthetic polymer, or a light or temperature activatablebiological polymer. The biological polymer may be configured to betemperature or light activatable to form a solidified polymer network.In some embodiments, the biological polymer may be modified toincorporate moieties providing the ability to be temperature or lightactivatable. The synthetic polymer modifications may include sizemodification motifs, cleavage motifs, reactive terminal moieties, and/orcell recognition motifs in any combination.

In some embodiments of the solidified polymer network of an isolationstructure, the solidified polymer network may include at least one of apolyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination. In other embodiments, the polymer may include at least oneof a polyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone(PCL), modified polycaprolactone, fibronectin, modified fibronectin,collagen, modified collagen, laminin, modified laminin, polysaccharide,modified polysaccharide, or a co-polymer in any combination. In yetother embodiments, the polymer may include at least one of apolyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin, ora co-polymer in any combination. In some embodiments, the solidifiedpolymer network does not include a silicone polymer. In someembodiments, the solidified polymer network may not include a polylacticacid (PLA) or a modified polylactic acid polymer. In other embodiments,the solidified polymer network may not include a polyglycolic acid (PGA)or a modified polyglycolic polymer. In some embodiments, the solidifiedpolymer network may not include a polyacrylamide or a modifiedpolyacrylamide polymer. In yet other embodiments, the solidified polymernetwork may not include a polyvinyl alcohol (PVA) or a modifiedpolyvinyl alcohol polymer. In some embodiments, the solidified polymernetwork may not include a polyacrylic (PAA) or modified PAA polymer. Insome other embodiments, the solidified polymer network may not include apolycaprolactone (PCL) or a modified polycaprolactone polymer. In otherembodiments, the solidified polymer network may not be formed from afibronectin or a modified fibronectin polymer. In some otherembodiments, the solidified polymer network may not be formed from acollagen or a modified collagen polymer. In some other embodiments, thesolidified polymer network may not be formed from a laminin or amodified laminin polymer.

Physical and chemical characteristics determining suitability of apolymer for use in the solidified polymer network may include molecularweight, hydrophobicity, solubility, rate of diffusion, viscosity (e.g.,of the medium), excitation and/or emission range (e.g., of fluorescentreagents immobilized therein), known background fluorescence,characteristics influencing polymerization, and pore size of asolidified polymer network. The solidified polymer network is formedupon polymerization or thermal gelling of a flowable polymer (e.g., apre-polymer solution,)

One type of polymer, amongst the many polymers that may be used, ispolyethylene glycol diacrylate (PEGDA). The mechanism of light initiatedpolymerization is shown in Equation 1. The free radical initiatorIgracure® 2959 (BASF), a highly efficient, non-yellowing radical, alphahydroxy ketone photoinitiator, is typically used for initiation atwavelengths in the UV region (e.g., 365 nm), but other initiators may beused. An example of another useful photoinitiator class forpolymerization reactions is the group of lithium acyl phosphinate salts,of which lithium phenyl 2, 4, 6,-trimethylbenzolylphosphinate hasparticular utility due to its more efficient absorption at longerwavelengths (e.g., 405 nm) than that of the alpha hydroxy ketone class.

Other types of PEG that may be photopolymerized include PEGdimethylacrylate, and/or multiarm PEG (n-PEG) acrylate (n-PEG-Acr).Other polymer classes that may be used include poly vinyl alcohol (PVA),polylactic acid (PLA) polyacrylic acid (PAA), polyacrylamide (PAM),polyglycolic acid (PGA) or polycaprolactone (PCL).

The molecular weight range of the polymer may be varied as required forthe performance of the isolation structures of the invention. A widerange of molecular weights of the flowable polymer may be suitable,depending upon the structure of the polymer. A useful star type polymermay have Mw (weight average molecular weight) in a range from about 500Da to about 20 kDa (e.g., four arm polymer), or up to about 5 kDa foreach arm or for a linear polymer, or any value therebetween. In someembodiments, a polymer having a higher molecular weight range, may beused at lower concentrations in the flowable polymer, and still providean in situ-generated barrier or isolation structure that may be used inthe methods described herein.

Various co-polymer classes may be used, including but not limited to:any of the above listed polymer, or biological polymers such asfibronectin, collagen or laminin. Polysaccharides such as dextran ormodified collagens may be used. Biological polymers havingphotoactivatable functionalities for polymerization may also be used.

Crosslinking may be performed by radiation of linear or branched PEGpolymers, free radical polymerization of PRG acrylates, and specificallytailored chemical reactions such as Michael addition, condensation,Click chemistry, native chemical ligation and/or enzymatic reactions.

The polymers may be selected to have a desired range of crosslinkingbased on the nature of the polymer (configuration of the flowablepolymers such as star, multiarm or comb polymers, length of polymersegments between crosslinkable functionalities) and polymerizationconditions (extent of temperature or photoinitiation, amount ofphotoactivatable initiator present, amount of radical terminator speciespresent, and the like).

In some embodiments, the polymer of the solidified polymer network maybe a modified PEG polymer. The polymer may be a star, 4-arm or 2-arm PEGdiacrylate polymer.

Swellable Polymers.

PEG polymers may be swellable under various conditions and may bereversed by reverting back to the original media/temperature.Poly-N-isopropylacrylamide (PNIPAm) may be swelled by increasingtemperature, and de-swelled by cooling.

Size Modification Motifs.

Some hydrogels, including poly-N-isopropylacrylamide (PNIPAm) or polyacrylamide (PAM), may also incorporate specific moieties such asazobenzene which changes cis/trans orientation upon exposure to light atthe surface of the functionalized polymer. This shift can providesignificant change in size of the portion of polymer such as anisolation structure within a pen. These polymers may alternativelyinclude cinnamic acid functionalities that cross link upon exposure toUV light, which is reversible upon removal of the light. Thecross-linked polymer is elongated compared to the non-crosslinked state.Another moiety which may be introduced to these polymers includestriphenyl leucomethane, which forms ion pairs upon application of light,reversibly, upon exposure to light. The wavelength of activating lightcan be brought into the visible range if trisodium copper chlorophyllinis incorporated into the polymer.

Other Modifications for Functionalization.

A polymer (e.g., PEG) may be modified by incorporating one or moredifferent motifs within the (PEG) polymer. The motifs may include sizemodification motifs, cleavage motifs, reactive terminal motifs, and/orcell recognition motifs, in any combination. A size modification motifmay include susceptibility to changes in temperature, ionic strength orpH of a surrounding medium that may cause a change in physical size ofthe solidified polymer network, thereby causing a change in size of theisolation structure. A non-limiting example may include a Lower CriticalSolution Temperature (LCST) or an Upper Critical Solution Temperature(UCST) polymer, such as poly N-isopropylacrylamide (PNIPAm). Anotherexample is the incorporation of disulfide bonds within a polymer such asPEB.

A cleavage motif may include a peptide sequence inserted into thepolymer that is a substrate for one or more proteases, including but notlimited to a matrix metalloproteinase, a collagenase, or a serineproteinase such as Proteinase K. Another category of cleavage motif mayinclude a photocleavable motif such as a nitrobenzyl photocleavablelinker which may be inserted into selected locations of the prepolymer.In some embodiments, a nitrobenzyl photocleavable linker may include a1-methinyl, 2-nitrobenzyl moiety configured to be photocleavable. Inother embodiments, the photocleavable linker may include a benzoinmoiety, a 1, 3 nitrophenolyl moiety, a coumarin-4-ylmethyl moiety or a1-hydroxy 2-cinnamoyl moiety. A cleavage motif may be utilized to removethe solidified polymer network of an isolation structure. In otherembodiments, the polymer may include cell recognition motifs includingbut not limited to a RGD peptide motif, which is recognized byintegrins.

Reversing/Removing/Minimizing the In Situ-Generated Isolation Structure.

A number of mechanisms may be used to remove or reduce the insitu-generated isolation structure when there is no further purpose forit. For example, once an assay is completed and desirable biologicalcells have been identified, it may be useful to remove the isolationstructure in order to continue culturing and expanding the biologicalcell demonstrating desirable activities or properties.

Mechanical force. Increasing flow can be used if at least a portion ofthe isolation structure is located within a flow region as opposed to anisolation region of a pen. For example, the at least one isolationstructure may be located within an isolation region of a sequestrationpen, and after the assay is complete, the sequestration pen or theisolation region therein may be modified to bring flow through theisolation region.

Hydrolytic susceptibility: Porogens, including polymers which areincapable of being chemically linked to the photoinitiated polymer(s),may be including when forming the isolation structure. The degree/sizeof openings within the formed hydrogel can customize the hydrolysis ratevia accessibility within the isolation structure). In other embodiments,the pores formed may be employed to permit secreted materials orchemical reagents to pass through the isolation structure but prevent acell from moving into, out of, and/or through the isolation structure.In other embodiments, degradability of these polymers may be increasedby introducing degradable segments such as polyester, acetal, fumarate,poly(propylene fumarate) or polyhydroxyacids into polymers (e.g., PEGpolymers).

Reducing agents: PEG may be formed with disulfide linkages at intervalsalong the macromere, which may be random or predetermined. The disulfidebonds may be broken by Dithiothreitol (DTT), mercaptoethanol, or TCEP.

Thermal: poly N-isopropylacrylamide (PNIPAm) or other suitable LCSTpolymers may be used to introduce isolation structures upon heating.They may be removed by decreasing the temperature of the formed polymerisolation structure. The polymers may include ELPs or other motifs thatalso permit removal by other mechanisms such as hydrolysis orproteolysis. In particular, PNIPAm may be used to create a surface foradherent cells, but then switched to permit export.

Proteolytic susceptibility: Hydrogels may have any sort of peptidesequence engineered in, such that selective proteolysis upon a selectedmotif by a selected protease can remove/reverse/or minimize a hydrogelisolation structure. Some classes of modified PEG include PEG havingelastin like peptide (ELP) motifs and/or having peptide motifs forsusceptibility to a variety of proteases (enzyme sensitive peptide ESP).A large number of these motifs are known. One useful motif is RGD whichmay be constrained to be cyclic.

Osmotic susceptibility: Calcium concentration/other osmotic strategiescan be employed to degrade and remove an isolation structure. As above,changes of media flowed through the channel or flow region maydimensionally swell or de-swell isolation structures.

Photocleavage: As described above, if a polymer of the solidifiedpolymer network includes a photocleavable moiety, directing illuminationof an exciting wavelength to the solidified polymer network will causecleavage within sections of the solidified polymer network. Thiscleavage may provide complete or partial disruption of the solidifiedpolymer network, thereby removing or reducing the isolation structure.If a partial disruption of the solidified polymer network is provided bythe photocleavage, complete disruption (e.g., complete removal of thisisolation structure) may be effected by flowing a fluidic medium in thechannel or flow region to sweep partially disrupted portions of thesolidified polymer network away from the isolated one or moremicro-objects.

In some applications, the isolation structure may not be removed but maysimply be swelled or de-swelled using light or media\solvent changes.Some types of hydrogels may incorporate moieties that respond reversiblyto light (for example, change regiochemistry about a rigid bond; formreversible crosslinks within the polymer, or form/break ion pairs).

Microfluidic (or Nanofluidic) Device Assisted Heating.

The microfluidic device may further include a metal pad disposed on thesubstrate at a location of the in situ-generated isolation structure.The metal pad may be created by deposing a contiguous metal shape or apattern of metal shapes onto the substrate. The thermal pad can compriseany type of metal that can be excited by a light source to produce heat.Suitable metals include chromium, gold, silver, aluminum, indium tinoxide, or any combination thereof. Metals may be combined in amulti-layered thermal pad, e.g., a layer of chromium, a layer oftitanium, a layer of gold. Other metals (and alloys) are known in theart. The thermal pad can comprise a continuous metal surface or cancomprise a pattern of metal (e.g. metal shapes such as dots, squares,lines, cones, irregular forms). In some embodiments, a gold pad may bedisposed on the substrate at a location where an in situ-generatedisolation structure will be/has been generated. The thermal pad may beused to generate heat to gel, swell, reduce, or remove an insitu-generated isolation structure. Heat may be generated by directinglight into the microfluidic device at the location where such gelling,swelling, reduction or removal is desired. In some embodiments, thesolidified polymer network may include a thermosensitive polymer. When asolidified polymer network of an isolation structure includes athermosensitive polymer, the device may further include a thermal paddisposed on the substrate at a location beneath the at least one insitu-generated isolation structure will be introduced.

Methods.

In situ-generated isolation structures, which may fully enclose a regionwithin the microfluidic device or partially enclose a region in themanner of a pen or sequestration pen, and which may be an insitu-generated barrier, may be introduced either before or afterintroduction of cells to the microfluidic (or nanofluidic) device. Thein situ-generated isolation structures may be designed to be temporaryor may be kept in place for at least the duration of a sorting and/orconcentrating and/or processing procedure.

The in situ-generated isolation structures may be introduced byphotoactivation, temperature change, or osmotic change which can cause apolymer solution present within the microfluidic to form an isolationstructure capable of preventing a biological cell or a bead fromcrossing the isolation structure. Depending on the mesh size of thepolymeric in situ-generated barrier/isolation structure, differentcategories of chemical species may be permitted to pass through thebarrier. If the mesh size is chosen to be about 2 nm, only smallmolecule components may be permitted to pass, but proteins, etc. maysequestered by the isolation structure/barrier. The in situ-generatedisolation structure/barrier may be formed of a polymer having a largermesh size that may not prevent smaller substances such as proteins,nucleic acids, organelles, or signaling molecules from crossing thebarrier. The in situ-generated isolation structure/barrier may permitmedia to pass through while not permitting a cell or a bead to enter,exit and/or pass through the in situ-generate structure/barrier. The insitu-generated isolation structure/barrier may have a mesh size(effective size of openings or voids between crosslinked polymerstrands) that permits a bead (including but not limited to a magneticbead, polystyrene bead or glass bead) to enter, exit and/or pass throughthe isolation structure/barrier while retaining a biological cell.

The process of introducing light activated polymerization can beperformed within the microfluidic device, and may additionally beperformed in the presence of cells. A photoactivatable polymerizationinitiator may be introduced before, contemporaneously, or after additionof the flowable polymer. Diffusion can compete with the polymerizationprocess, so the ability to quickly create free radicals may be useful.Additionally, free radicals can quickly combine with free oxygen. Whilephotopolymerization may be very efficient and quick in the absence ofoxygen in the media, when biological cells are present (thus requiringthe presence of oxygen), adjustments to the number of initiatingradicals may be made to compensate. In fact, for the introduction ofmany of the types of barriers useful within a microfluidic device, thelimiting effect of oxygen may be helpful as chain termination may happenmore quickly and may limit the amount of extraneous polymer formed,particularly when introducing limited amounts of polymer to form smallbarriers that do not entirely block a pen or a channel.

In the methods of isolating micro-objects with in situ generatedisolation structures, including any variations described herein, amicrofluidic environment is provided where micro-objects may be sorted,concentrated, and/or selectively disposed in preselected regions orsequestration pens of a microfluidic device. Micro-objects isolated bythe in situ-generated isolation structures described herein may beselectively retained within the microfluidic device while non-isolatedmicro-objects are exported, and may further be selectively releasedthereafter for further processing in the absence of the micro-objectsnot selected by the isolation process. Additionally, either isolatedmicro-objects or non-isolated micro-objects may further be selectivelyprocessed in the microfluidic device, which may include any type ofassay or preparation for further processing such as lysis, gene editingor genotyping.

In one aspect, a method of isolating a micro-object in a microfluidicdevice is provided, including the steps of: providing the microfluidicdevice, where the microfluidic device includes an enclosure including asubstrate and a flow region; introducing a first fluidic medium into theenclosure of the microfluidic device; introducing a plurality ofmicro-objects in the fluidic medium into the enclosure; introducing aflowable polymer into the enclosure before or after the introducing ofthe plurality of micro-objects; activating solidification of theflowable polymer at at least one selected area of the flow region,thereby forming an in situ-generated isolation structure; and isolatingat least one of the plurality of micro-objects with the insitu-generated isolation structure.

In various embodiments of the method, the method may further includeexporting a remainder of the plurality of micro-objects from themicrofluidic device. The remainder of the plurality of micro-objects maybe a selected portion of the plurality of micro-objects. In otherembodiments, the at least one micro-object isolated by the insitu-generated isolation structure may be the selected portion of theplurality of micro-objects.

In various embodiments of the method, the method may further include thestep of reducing or removing the in situ-generated isolation structure(which may include an in situ-generated barrier, one or more insitu-generated barrier modules, and/or one or more in situ-generatedisolation modules) by increasing flow of a fluidic medium, introducing ahydrolytic agent, introducing a proteolytic agent, increasing/decreasingosmolality of the fluidic medium, changing temperature of the insitu-generated isolation structure, or optically illuminating the insitu-generated isolation structure, thereby releasing the at least onemicro-object from being isolated. The step of changing the temperaturemay further include optically illuminating a thermal pad on thesubstrate adjacent to or under the in situ-generated isolationstructure. In various embodiments of the method, the method may furtherinclude the step of exporting the at least one released micro-objectfrom the microfluidic device. In various embodiments of the method,exporting the selected portion of the plurality of micro-objects fromthe microfluidic device may further include moving the selected portionof the plurality of micro-objects to a different portion of thesubstrate of the microfluidic device.

In various embodiments of the method, the in situ-generated isolationstructure may be porous to a flow of a fluidic medium, while preventingsome or all subsets of micro-objects from entering, exiting and/orpassing through the in situ-generated isolation structure.

In various embodiments of the method, the step of activatingsolidification of the flowable polymer may form an in situ-generatedisolation structure that may be an in situ-generated pen. FIG. 9C showsone non-limiting example. The in situ-generated pen may be selected fromthe group consisting of: an in situ-generated sequestration penincluding an isolation region and a connection region, the connectionregion having a proximal opening to the flow region and a distal openingto the isolation region; an in situ-generated wall completely enclosingthe at least one micro-object; and an in situ-generated pen partiallyenclosing the at least one micro-object where the in situ-generated penhas one opening in its periphery sufficiently large to permitentrance/exit of at least one micro-object. In some embodiments, themethod may further include the step of swelling the in situ-generatedpen around the at least one isolated micro-object, thereby applyingpressure to and permeabilizing the micro-object. FIG. 20A shows anon-limiting example.

The step of activating solidification of the flowable polymer mayfurther include forming an in situ-generated pen having a plurality ofin situ-generated pen modules, each of the plurality of sub-units spacedapart from each other at a distance preventing the at least one isolatedmicro-object from exiting the pen. In some embodiments, the plurality ofin situ-generated pen modules may be spaced apart from each other at adistance preventing at least one subset of micro-objects of theplurality of micro-objects from passing through the in situ-generatedpen. The micro-objects may have a diameter between about 1-20 microns.In some embodiments, the at least one subset of micro-objects includesat least one type of biological cell.

In some embodiments, the step of activating solidification of theflowable polymer may form an in situ-generated isolation structureincluding an in situ-generated barrier configured to prevent the atleast one micro-object from passing through the barrier. In someembodiments, a plurality of barriers may be introduced, and theplurality of barriers may be configured to allow isolation of respectivesub-sets of at least one micro-object of the plurality of micro-objects.The method may include a step of reducing a size or removing the in-situgenerated isolation structure, thereby releasing the at least onemicro-object.

In various embodiments of the method, the method may further include thestep of exporting the one or more sub-sets of the at least one releasedmicro-object from the microfluidic device. Exporting the one or moresub-sets of the at least one micro-object may include moving the one ormore sub-sets of the at least one micro-object to a different portion ofthe microfluidic device. The other portion of the microfluidic devicemay include a sequestration pen.

In various embodiments of the method, the step of activatingsolidification may further include forming an in situ-generated barrierhaving a plurality of in situ-generated barrier modules, each of theplurality of in situ-generated modules spaced apart from each other at adistance preventing the at least one micro-object from passing throughthe in situ-generated barrier. In various embodiments of the method, themethod may further include the step of spacing the plurality of insitu-generated barrier modules apart from each other thereby forming anin situ-generated barrier preventing at least one subset ofmicro-objects of the plurality of micro-objects from passing through thein situ-generated barrier. The at least one subset of micro-objects mayinclude at least one type of biological cell.

In various embodiments of the method, the enclosure of the microfluidicdevice may further include at least one sequestration pen including anisolation region and a connection region, the connection region having aproximal opening to the flow region and a distal opening to theisolation region. The enclosure may include a plurality of sequestrationpens. The plurality of sequestration pens may be aligned in a row, andthe proximal opening of each of the plurality of sequestration pens maybe disposed contiguously to each other. In some embodiments, the flowregion may include a channel and the proximal opening of each of theplurality of sequestration pens may open off one side of the channel.

In various embodiments of the method, the step of activation ofsolidification may be performed inside a sequestration pen. In someembodiments, the step of activating solidification may be performedwithin the isolation region or the connection region. FIGS. 4, 10A-C,12B, 16, 19, 21A-C and 22 show non-limiting examples. In someembodiments, the step of activating solidification within thesequestration pen may form an in situ-generated pen. The insitu-generated pen may encompass a single biological micro-object. Insome embodiments, the method may further include the step of swellingthe in situ-generated pen thereby more tightly surrounding the singlebiological cell. The method may further include the step of swelling thein situ-generated pen until the single biological cell is permeabilized.FIG. 20A shows one non-limiting example. In various embodiments of themethod, the step of activating solidification of the flowable polymerwithin the sequestration pen may form an in situ-generated barrier. Insome embodiments, the in situ-generated barrier may be disposed in theisolation region of the sequestration pen. The in situ-generated barriermay have a width across the isolation region that may be about ¼ toabout ¾ of a width of the isolation region. The width of the insitu-generated barrier across the isolation region may be about 5microns to about 20 microns. The width of the isolation region may beabout 30 microns to about 50 microns. FIGS. 12A and B shows onenon-limiting example. The method may include the step of exporting aremainder of the plurality of micro-objects not isolated by the barrierfrom the microfluidic device. In various embodiments of the method, themethod may further include the step of reducing or removing the insitu-generated barrier by optically illuminating or changing atemperature around the in situ-generated barrier, thereby releasing theat least one micro-object. In various embodiments of the method, themethod may further include the step of exporting the at least onemicro-object from the microfluidic device.

In some embodiments, the step of activating solidification of theflowable polymer may generate an in situ-generated barrier in theconnection region. The method may further include the step of modifyingthe in situ-generated barrier to include a capture moiety configured tocapture at least one micro-object disposed in the sequestration pen. Thein situ-generated barrier may have a width across the connection regionthat may be about ¼ to about ¾ of a width of the connection region. Insome embodiments, the width of the in situ-generated barrier across theconnection region may be about 5 microns to about 20 microns. FIGS.10A-C show one non-limiting example. In various embodiments, theconnection region may have a width of about 30 microns to about 5microns.

In some embodiments, the method may further include the step ofexporting a remainder of the plurality of micro-objects not isolated bythe in situ-generated barrier from the microfluidic device.

In various embodiments of the method, the method may further include astep of reducing or removing one or more of the plurality of insitu-generated barriers, thereby releasing the at least one micro-objectfrom isolation. In various embodiments, the method may further includethe step of exporting the at least one micro-object from themicrofluidic device, after it has been released from isolation by the insitu-generated barrier.

In some embodiments, the step of activating solidification of theflowable polymer may form an in situ-generated barrier in the channel.In various embodiments, the step of activating solidification of theflowable polymer may dispose the in situ-generated barrier adjacent to aproximal opening of at least one sequestration pen of the plurality ofsequestration pens. In some embodiments, where a plurality ofsequestration pens may be present and the plurality of sequestrationpens form a row, and the step of activating solidification of theflowable polymer may dispose the in situ-generated barrier adjacent to adistal edge of the proximal opening of a sequestration pen located atthe end of the row of sequestration pens. In some embodiments of themethod, the isolating step may include preventing the at least onemicro-object of the plurality of micro-objects from moving past the insitu-generated barrier in the channel. In other embodiments, theisolating step may include preventing at least one subset of theplurality of micro-objects from moving past the in situ-generatedbarrier in the channel. FIGS. 6, 7, 8, and 13A-C show non-limitingexamples.

In various embodiments of the method, the step of activatingsolidification of the flowable polymer may further include forming an insitu-generated barrier having a plurality of in situ-generated barriermodules, each of the plurality of modules spaced apart from each otherat a distance preventing the at least one micro-object of the pluralityof micro-objects from moving past the barrier in the channel. In someembodiments, the step activating solidification of the flowable polymermay further include forming the plurality of in situ-generated barriermodules at a distance preventing at least one subset of micro-objects ofthe plurality of micro-objects from moving past the barrier. In someembodiments, where a plurality of sequestration pens may be present andthe plurality of sequestration pens form a row, and the step ofactivating solidification of the flowable polymer may dispose thebarrier adjacent to a distal edge of the proximal opening of asequestration pen located at the end of the row of sequestration pens.In various embodiments, the isolating step may include preventing the atleast one micro-object from passing the selected sequestration pen.FIGS. 9A-D shows a non-limiting example. In some embodiments, theisolating step may further include disposing the at least onemicro-object into the selected sequestration pen. The at least onemicro-object may be all of the plurality of micro-objects.

In some embodiments, the step of activating solidification of theflowable polymer may form an in situ-generated barrier sized to blockthe proximal openings of at least two contiguous sequestration pens. Thein situ-generated barrier blocking the proximal openings may have adimension of at least 50 microns to about 500 microns. FIGS. 5A and Bshow one non-limiting example. In various embodiments, the method mayfurther include the step of exporting a remainder of a remainder of theplurality of micro-objects not isolated by the barrier from themicrofluidic device.

In some embodiments, the step of activating solidification of theflowable polymer may form the barrier at a distal edge of the proximalopening of a first sequestration pen of the plurality of pens. FIGS.13A-C shows one non-limiting example. In some embodiments, the pluralityof sequestration pens may be a first plurality of sequestration pens andthe channel may be a first channel, and the microfluidic device mayfurther include a second plurality of sequestration pens disposed alonga second channel, and the barrier may prevent at least one micro-objectfrom passing the in situ-generated barrier into the first channel. Thebarrier may direct flow of fluidic medium to another portion of theenclosure. In some embodiments, the barrier may prevent all of theplurality of micro-objects from passing the barrier into the firstchannel. In some embodiments, the isolating step may further includedirecting the plurality of micro-objects into the second channel. Insome embodiments, the isolating step may further include disposing theplurality of micro-objects into the second plurality of sequestrationpens in the second channel.

In various embodiments of the method, the microfluidic device mayinclude a first plurality of sequestration pens disposed adjacent toeach other on a first side of the channel and the microfluidic devicefurther includes a second plurality of sequestration pens disposedadjacent to each other on a second opposing side of the microfluidicchannel. FIGS. 14A-B and 15 show non-limiting examples. In someembodiments, the step of introducing the plurality of micro-objects mayinclude introducing a plurality of micro-objects to each of the firstplurality of sequestration pens in a first fluidic medium. Each of theplurality of micro-objects may be a clonal population of biologicalmicro-objects. In various embodiments of the method, the step ofintroducing may further include introducing a first biologicalmicro-object of the first clonal population to a first sequestration penof the second plurality of sequestration pens in the first fluidicmedium. In some embodiments, the step of introducing may further includeintroducing a first biological micro-object of each respective clonalpopulation in each sequestration pen of the first plurality ofsequestration pens to a respective sequestration pen in the secondplurality of sequestration pens in the first fluidic medium.

In some embodiments, the step of activating solidification of theflowable polymer may include activating solidification of the flowablepolymer along a length of the microfluidic channel, thereby forming anin situ-generated isolation structure including an in situ-generatedbarrier dividing the microfluidic channel into a first sub-channelconfigured to provide a first sub-flow of a fluidic medium past thefirst plurality of sequestration pens and a second sub-channelconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of pens, wherein the in situ-generated barrier preventsmicro-objects from moving from the first sub-channel to the secondsub-channel, and vice versa.

In other embodiments, the microfluidic device may further include abarrier dividing the microfluidic channel into a first sub-channelconfigured to provide a first sub-flow of a fluidic medium past thefirst plurality of sequestration pens and a second sub-channelconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of pens, the barrier punctuated by at least one gapaligned with a proximal opening to the first sub-channel of a firstsequestration pen of the first plurality of sequestration pens andaligned with a proximal opening to a proximal opening to the secondsub-channel of a first pen of the second plurality of sequestrationpens; and further wherein the step of activating polymerization mayinclude activating polymerization at the at least one gap to form atleast one in situ-generated barrier in the at least one gap, therebypreventing micro-objects from moving from the first sub-channel to thesecond sub-channel, and vice versa. In some embodiments, when themicrofluidic device includes a barrier punctuated by at least one gap,the step of moving the at least one cell of each clonal population inthe first plurality of sequestration pens comprises moving the at leastone cell to the respective sequestration pen of the second plurality ofpens through the at least one gap. In some embodiments, the barrier hasa plurality of gaps. Each gap may be aligned across from the proximalopening (to the microfluidic channel) of each sequestration pen of thefirst plurality of sequestration pens and, optionally, also aligned withthe proximal opening of each respective sequestration pen of the secondplurality of sequestration pens to the microfluidic channel. The step ofactivating solidification can include forming a one or more insitu-generated barriers closing the one or more of the plurality ofgaps. In various embodiments of the method, the method may furtherinclude the step of introducing a second fluidic medium into the secondsub-channel. The method may further include the step of flowing thefirst fluidic medium in the first sub-channel and flowing the secondfluidic medium in the second sub-channel along the in situ-generatedbarrier to respective first and second outputs of the device. In someembodiments, the isolating step may further include preventing the firstmedium from mixing with the second medium in the second sub-channel. Inyet further embodiments, the isolating step may further include growingeach of the first biological micro-objects of each respective clonalpopulations in the second fluidic medium, whereby the first biologicalmicro-objects in the second plurality of sequestration pens growdifferently from the respective clonal populations in the firstplurality of sequestration pens. The method may further include the stepof expanding each of the first biological micro-objects in each of thesecond plurality of sequestration pens to provide new clonal populationsof biological micro-objects. In various embodiments, the step ofactivating solidification of the flowable polymer may further includeforming a barrier having a plurality of in situ-generated barriermodules, each of the plurality of modules spaced apart from each otherat a distance preventing the at least one micro-object from passingthrough the barrier. The step of activating solidification of theflowable polymer may further include spacing the plurality of insitu-generated barrier modules apart from each other thereby forming anin situ-generated barrier preventing at least one subset ofmicro-objects of the plurality of micro-objects from passing through thebarrier. The in situ-generated barrier has a length along themicrofluidic channel. In some embodiments, the step of activatingsolidification of the flowable polymer may further include forming afirst module of the plurality of in situ-generated modules having alength of at least 40% of the length of the in situ-generated barrier.FIG. 15 shows one non-limiting example. In other embodiments, the stepof activating solidification of the flowable polymer may further includeforming each of a remainder of the plurality of in situ-generatedbarrier modules having a length no greater than 20% of the length of thebarrier. The in situ-generated barrier may have a first upstream (e.g.,closer to a fluidic inlet) end and a second downstream end (e.g., closerto a fluidic outlet), and a length there between. In some embodiments,the step of activating solidification of the flowable polymer mayfurther include forming the barrier, where the in situ-generated barriermay not be porous to a flow of the first or second fluidic medium at thefirst end and may be porous to at least a portion of the flow of thefirst or the second fluidic medium at a point that is at least 40% ofthe length of the in situ-generated barrier.

In various embodiments of the method, the method may further include thestep of reducing a dimension of or removing the in situ-generatedbarrier. In some embodiments, when the in situ-generated barrierincludes a plurality of modules, the in situ-generated barrier may bereduced by removing at least a portion of the plurality of barriermodules. Fewer or more of the in situ-generated barrier modules may beremoved to reduce the barrier. In various embodiments of the method, themethod may further include the step of growing each of the firstbiological micro-objects of each respective clonal populations in adiffering composition of fluidic medium depending on an extent of thefirst fluidic medium and the second fluidic medium mixing.

In any of the embodiments of the method, the method may include a stepof processing an isolated micro-object. Alternatively, the method mayinclude a step of processing a micro-object that has not been isolated,while another micro-object in the microfluidic device is isolated.

In another aspect, a method of isolating a micro-object in amicrofluidic device is provided, including the steps of: providing amicrofluidic device including an enclosure having a substrate, a flowregion including a channel, and a plurality of sequestration pens;disposing a fluidic medium including a plurality of micro-objects intothe channel of the microfluidic device, where the fluidic mediumincludes a flowable polymer; disposing select micro-objects of theplurality of micro-objects in at least a portion of the plurality ofsequestration pens thereby forming a plurality of populatedsequestration pens, each containing at least one micro-object; selectingat least one of the plurality of populated sequestration pens;initiating polymerization of the flowable polymer at a selected pointwithin the connection region, the isolation region, or at the proximalopening of the connection region of the at least one selectedsequestration pen, where the polymerized polymer of the flowable polymergenerates at least a partial in situ-generated barrier; and prevents theat least one micro-object from exiting the at least one selectedsequestration pen. FIGS. 4 and 5 are nonlimiting examples of thismethod. Each of the plurality of sequestration pens may include anisolation region and a connection region, the connection region having aproximal opening to the channel and a distal opening to the isolationregion. In some embodiments, polymerization of the flowable polymerforms a solidified polymer network. In various embodiments, the methodmay further include the step of removing at least one of the pluralityof micro-objects from one or more unselected populated sequestrationpens. The method may further include the step of subsequently permittingthe at least one micro-object to exit the at least one selectedsequestration pen.

The method may further include the step of permitting the at least onemicro-object to exit the at least one selected sequestration pen furtherincludes at least reducing the at least partial in situ-generatedbarrier, thereby releasing the at least one micro-object. Reducing an insitu-generated barrier can include shrinking a size of the insitu-generated barrier or can include shifting the in situ-generatedbarrier into a second state that has a smaller size. Reducing may alsoinclude increasing porosity of the remaining portion(s) of the barrier.Reducing may further include removing a portion of the modules of an insitu-generated barrier such that the reduced in situ-generated barriermay permit more types of micro-objects to pass in and out through thereduced in situ-generated barrier. In some embodiments, the method mayfurther include the step of reducing or removing the at least partial insitu-generated barrier by increasing flow of a fluidic medium,introducing a hydrolytic agent, introducing a proteolytic agent,increasing/decreasing osmolality of the fluidic medium, changingtemperature of the at least partial barrier, or optically illuminatingthe in situ-generated barrier, thereby releasing the at least onemicro-object from isolation. The step of changing temperature mayfurther include optically illuminating a thermal pad on the substrateadjacent or under the barrier. In some embodiments, the step of formingan in situ-generated barrier may further include forming a substantiallycomplete in situ-generated barrier. In various embodiments, thesubstantially complete in situ-generated barrier may be formed at theproximal opening to the channel of the at least one sequestration pen.The forming step may further include forming the substantially completein situ-generated barrier at the proximal opening of more than onesequestration pen. In some embodiments, the enclosure of themicrofluidic device may further include a plurality of contiguoussequestration pens. In various embodiments, the substantially completein situ-generated barrier may be removed by an increased flow of fluidicmedium in the channel.

Directing Fluidic Flow which May Include Micro-Objects.

In another aspect, a method of directing fluidic flow within amicrofluidic device is provided, including the steps of: providing amicrofluidic device including an enclosure having a substrate and a flowregion configured to contain a fluidic medium that divides into at leasta first flow region and a second flow region; generating an insitu-generated barrier in situ that blocks fluid flow into the firstflow region; introducing the fluidic medium into the flow region; andflowing the fluidic medium through the enclosure, such that the insitu-generated barrier directs the flow of the fluidic medium throughthe second flow region. In various embodiments, the fluidic medium mayinclude at least one micro-object. In some embodiments, the at least onemicro-object may be directed into the second flow region. FIGS. 13A-Cshow one nonlimiting example of this method.

In various embodiments of the method, the method may further include thestep of disposing the at least one micro-object within a sequestrationpen disposed within the second flow region. The sequestration pen mayinclude an isolation region and a connection region, the connectionregion having a proximal opening to the second flow region and a distalopening to the isolation region. The at least first micro-object may bedisposed within the isolation region of the sequestration pen.

In various embodiments of the method, the method may further include thestep of removing the in situ-generated barrier to substantially unblockthe first flow region. The step of substantially unblocking the firstportion of the flow region may further include allowing flow of thefluidic medium containing a micro-object into the first flow region. Insome embodiments, at least a portion of the in situ-generated barriermay be removable by application of increased flow in the flow region,hydrolysis, proteolysis, osmotic change, temperature change of thebarrier, or optical illumination.

In various embodiments of the method, the method may further include thestep of introducing a second in situ-generated barrier configured toblock the second flow region.

In some embodiments, the method may further include the step ofintroducing at least one micro-object into the first flow region. Themethod may further include the step of flowing the fluidic mediumthrough the flow region, thereby directing the at least one micro-objectto the first flow region. In some embodiments, the directing step mayfurther include disposing the at least one micro-object within asequestration pen disposed within the first flow region. Thesequestration pen may include an isolation region and a connectionregion, the connection region having a proximal opening to the flowregion and a distal opening to the isolation region, and further wherethe at least one micro-object is disposed within the isolation region ofthe sequestration pen. In various embodiments of the method, the methodmay further include the step of removing the second in situ-generatedbarrier.

In various embodiments of the method, the method may further include thestep of processing the at least one micro-object. The further processingstep may include assaying, sorting, permeabilizing, transfecting orexporting the at least one micro-object.

In various embodiments, the generating step may include initiatingsolidification of a flowable polymer present in the fluidic medium.

In various embodiments of the method, the at least first micro-objectand/or the at least second micro-object may be a biologicalmicro-object.

Concentrating micro-objects, which may include concentrating biologicalcells.

In another aspect, a method of concentrating micro-objects in amicrofluidic device may be provided, including the steps of: providing amicrofluidic device including an enclosure having a substrate and a flowregion configured to contain a fluidic medium; introducing an insitu-generated isolation structure in a first sector of the flow region,where the in situ-generated isolation structure is configured to permitthe fluidic medium to flow through the in situ-generated isolationstructure but does not permit at least one micro-object in the fluidicmedium to pass through the isolation structure; introducing a firstplurality of micro-objects in a first volume of the fluidic medium intothe first sector of the flow region; and concentrating at least a firstsubset of the first plurality of micro-objects in the first sector ofthe flow region. FIGS. 6, 7, 8A-B, 9A-D, 10A-C show some embodiments ofthe method. In various embodiments, the first volume of the fluidicmedium may be larger than a volume of the first sector of the flowregion.

In some embodiments, the in situ-generated isolation structure may notpermit a first subset of the first plurality of micro-objects to flowthrough the isolation structure but may permit a second subset of thefirst plurality of micro-objects to flow through the in situ-generatedisolation structure. In some embodiments, the step of concentrating theat least first subset of the first plurality of micro-objects in thefirst sector may further include sorting the second subset ofmicro-objects from the first subset of micro-objects of the firstplurality of micro-objects.

In various embodiments of the method, the method may further include thestep of disposing the at least first subset of the first plurality ofmicro-objects within at least one sequestration pen located within thefirst sector. Each sequestration pen may include an isolation region anda connection region, the connection region having a proximal opening tothe flow region and a distal opening to the isolation region.

In various embodiments of the method, the method may further include thestep of introducing a second plurality of micro-objects into the flowregion, and flowing the second volume of fluidic medium through thefirst sector of the flow region; and concentrating at least a firstsubset of the second plurality of micro-objects along with the at leastfirst subset of the first plurality in the first sector of the flowregion. The method may further include the step of disposing the atleast first subset of the first plurality and the at least first subsetof the second plurality of micro-objects within at least onesequestration pen located within the first sector.

In various embodiments of the method, the method may further include thestep of introducing a second plurality of micro-objects in a secondvolume of fluidic medium into the first sector of the flow region,thereby sorting a first subset of micro-objects from a second subset ofmicro-objects of the second plurality of micro-objects and concentratingthe at least first subset of the first plurality of micro-objects andthe at least first subset of the second plurality of micro-objects inthe first sector of the substrate.

In some embodiments, the method may further include the step ofdisposing the at least first subset of the first plurality ofmicro-objects and the first subset of the second plurality ofmicro-objects into at least one sequestration pen located within thefirst sector, where each sequestration pen includes an isolation regionand a connection region, the connection region having a proximal openingto the flow region and a distal opening to the isolation region.

In various embodiments of the method, the method may further include thestep of removing or reducing at least a portion of the isolationstructure, thereby permitting a volume of fluidic medium containingmicro-objects to flow to at least a second sector of the flow region. Invarious embodiments of the method, the method may further include thestep of introducing a second in situ-generated isolation structure inthe second sector of the flow region. In some embodiments of the method,the method may further include the step of introducing a secondplurality of micro-objects in a second volume of fluidic medium into thefirst sector of the flow region; and concentrating the at least firstsubset of the plurality of micro-objects within the second sector of theflow region. The method may further include the step of disposing the atleast first subset of the second plurality of micro-objects within atleast one sequestration pen located within the second sector of the flowregion. The method may further include the step of removing or reducingat least a portion of the second isolation structure, thereby permittingunrestricted flow throughout the flow region.

In various embodiments of the method, the in situ-generated isolationstructure may be a fully enclosed pen encompassing a selectedmicro-object, a pen open at a portion of its periphery large enough toadmit passage of a micro-object, a sequestration pen including anisolation region and a connection region, the connection region having aproximal opening to the flow region and a distal opening to theisolation region, or a barrier. In some embodiments, the insitu-generated barrier may include a plurality of in situ-generatedbarrier modules, each spaced from the remainder of the plurality suchthat a size of an opening between two in situ-generated modules issmaller than a size of a selected micro-object. The micro-objects mayhave a diameter from between about 1-20 μm.

In some embodiments, the in situ-generated isolation structure may be anin situ-generated barrier and may be substantially disposed within theisolation region or connection region of the sequestration pen. In otherembodiments, the in situ-generated isolation structure may be an insitu-generated barrier and may be substantially disposed within the flowregion.

In various embodiments, the first plurality of micro-objects may includea biological micro-object. In some embodiments, the first subset and thesecond subset of the first plurality of micro-objects may includedifferent types of micro-objects. In various embodiments, the secondplurality of micro-objects may include a biological micro-object. Whenboth pluralities of micro-objects are biological micro-objects, thefirst subset and the second subset of the second plurality ofmicro-objects may include different types of micro-objects.

Assaying Cells of a Clonal Population.

In another aspect, a method of assaying cells of a clonal population ina microfluidic device is provided; and includes the steps of:introducing a first fluidic medium including a plurality of cells intoan enclosure of the microfluidic device, the enclosure including asubstrate, a flow region including a microfluidic channel configured tocontain a fluidic medium, a first plurality of sequestration pensdisposed adjacent to each other such that each sequestration pen of thefirst plurality opens off a first side of the microfluidic channel, anda second plurality of sequestration pens disposed adjacent to each othersuch that each sequestration pen of the second plurality opens off asecond opposing side of the microfluidic channel; flowing the firstfluidic medium and the plurality of cells into the channel of themicrofluidic device; introducing a clonal population of cells in each ofthe sequestration pens of the first plurality of sequestration pens; foreach clonal population of cells in the first plurality of sequestrationpens, moving at least one cell to a respective sequestration pen of thesecond plurality of sequestration pens; introducing a flowable polymerinto the channel; activating solidification of the flowable polymeralong a length of the microfluidic channel, thereby forming an insitu-generated barrier dividing the microfluidic channel into a firstsub-channel configured to provide a first sub-flow of fluidic mediumpast the first plurality of sequestration pens and a second sub-channelconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of sequestration pens, wherein the in situ-generatedbarrier prevents cells from moving from the first sub-channel to thesecond sub-channel, and vice versa; flowing a second fluidic medium intothe second sub-channel, wherein the second fluidic medium includesreagents for assaying the cells in the second plurality of sequestrationpens; and assaying the cell(s) in each sequestration pen of the secondplurality. FIGS. 14A-C show one embodiment. In various embodiments, thein situ-generated barrier may have a length from a first end of thechannel to a second end of the microfluidic channel. Each sequestrationpen of the first plurality of sequestration pens and of the secondplurality of sequestration pens may have a proximal opening to itsrespective side of the microfluidic channel.

In various embodiments, the flowable polymer may be introduced before orafter the step of introducing of the plurality of cells to the firstplurality of sequestration pens.

In some embodiments, the step of introducing the clonal population mayinclude introducing a single cell to each of the first plurality ofsequestration pens, and may further include expanding the single cell toa clonal population of cells.

In various embodiments of the method, the method may further include thestep of flowing fluidic medium in the first sub-channel and flowingfluidic medium in the second sub-channel to respective first and secondoutputs of the microfluidic device. The fluidic medium in the firstsub-channel may be different from the fluidic medium in the secondsub-channel. In some embodiments, the in situ-generated barrier mayprevent the first sub-flow of fluidic medium in the first sub-channelfrom mixing with the second sub-flow of fluidic medium in the secondsub-channel.

In some embodiments, the step of assaying may include preparing thecells in the second plurality of sequestration pens for genotyping. Insome other embodiments, the step of assaying may include determining alevel of production of a biological product by the cell(s) in eachsequestration pen of the second plurality and/or by the clonalpopulation in each sequestration pen of the first plurality.

In some embodiments, the reagents for assaying may include one or moreof the group including chemical reagents, biological reagents, feedercells, stimulatory cells, reporter cells, reporter molecules, and beads.The beads may include chemical reagents, biological reagents,stimulatory reagents, or reporter molecules.

In various embodiments, the assaying step further includes identifyingat least one cell of the cells in the second plurality of sequestrationpens, the at least one cell including a selected characteristic. Themethod may further include a step of exporting at least one cell of thecells in the second plurality of sequestration pens, wherein the atleast one cell includes a selected characteristic s. In variousembodiments, the method may further include the step of exporting theclonal population from the respective pen of the first plurality ofsequestration pens. In other embodiments, the method may further includea step of exporting the cells in the second plurality of sequestrationpens and/or clonal populations of cells in the first plurality ofsequestration pens that do not include a selected characteristic.

In various embodiments of the method, the method may further include thestep of removing the in situ-generated barrier before exporting a cell.

In various embodiments, the step of activating solidification of theflowable polymer along a length of the microfluidic channel includesactivating solidification of the flowable polymer at gaps of a barrierextending from a first end of the microfluidic channel to a second endof the microfluidic channel, the barrier separating the microfluidicchannel into the first sub-channel and the second sub-channel, whereinthe gaps are aligned with the proximal opening of each pen of the firstplurality of sequestration pens and the proximal opening of therespective pen of the second plurality of sequestration pens, therebyforming an in situ-generated barrier preventing cells from moving fromthe first sub-channel to the second sub-channel, and vice versa. Thebarrier may have at least one gap aligned with the proximal opening ofone pen of the first plurality of sequestration pens and the proximalopening of the respective pen of the second plurality of sequestrationpens. In some embodiments, the barrier has a plurality of gaps. Each gapmay be aligned across from the proximal opening (to the microfluidicchannel) of each sequestration pen of the first plurality ofsequestration pens and, optionally, also aligned with the proximalopening of each respective sequestration pen of the second plurality ofsequestration pens to the microfluidic channel. In some embodiments, thestep of activating solidification along the length of the barrier mayinclude forming a plurality of in situ-generated barriers closing theplurality of gaps in the barrier.

Cell Line Evolution.

In another aspect, a method of cell line evolution in a microfluidicdevice is provided, including the steps of: providing the microfluidicdevice, where the device includes an enclosure comprising a substrate, aflow region including a channel configured to contain a fluidic medium,a first plurality of sequestration pens disposed adjacent to each othersuch that each sequestration pen of the first plurality opens off afirst side of the microfluidic channel, and a second plurality ofsequestration pens disposed adjacent to each other such that eachsequestration pen of the second plurality opens off a second opposingside of the microfluidic channel; introducing a first fluidic mediuminto the channel of the microfluidic device; introducing one or morecells of a clonal population into each of the first plurality ofsequestration pens; introducing one or more cells of the clonalpopulation into each of the second plurality of sequestration pens;introducing a flowable polymer into the channel; activatingsolidification of the flowable polymer along a length of themicrofluidic channel, thereby forming an in situ-generated barrierdividing the microfluidic channel into a first sub-channel configured toprovide a first sub-flow of fluidic medium past the first plurality ofsequestration pens and a second sub-channel configured to provide asecond sub-flow of fluidic medium past the second plurality ofsequestration pens, wherein the in situ-generated barrier prevents cellsfrom moving from the first sub-channel to the second sub-channel, andvice versa; introducing a second fluidic medium into the second side ofthe channel; and growing each of the one or more cells in eachsequestration pen of the second plurality of sequestration pens in thepresence of the second fluidic medium. The step of introducing aflowable polymer may be performed before or after the introducing ofcells.

In some embodiments, the second fluidic medium may include chemical orbiological components differing from those of the first fluidic medium.The chemical or biological components of the second fluidic medium mayapply selective pressure upon the growth of the one or more cells of theclonal population in each of the second plurality of sequestration pens.

In various embodiments of the method, the method may further include thestep of flowing the first fluidic medium in the first side of thechannel and flowing the second fluidic medium in the second side of thechannel along the in situ-generated barrier to respective first andsecond outputs of the device. In some embodiments, the isolating stepmay further include preventing the first medium from mixing with thesecond medium on the second side of the channel.

In various embodiments of the method, the method may further include thestep of expanding each of the single cells in each of the secondplurality of sequestration pens to provide new clonal populations ineach sequestration pen of the second plurality of sequestration pens.

In some embodiments, the step of forming an in situ-generated barriermay further include forming an in situ-generated barrier having aplurality of in situ-generated barrier modules, each of the plurality ofin situ-generated modules spaced apart from each other at a distancepreventing the at least one micro-object from passing through thebarrier. In some embodiments, the micro-objects prevented from passingthrough the in situ-generated barrier may have a diameter from 1-20microns. In other embodiments, the step of forming an in situ-generatedbarrier may further include spacing the plurality of in situ-generatedbarrier modules apart from each other thereby forming an insitu-generated barrier preventing at least one subset of micro-objectsof the plurality of micro-objects from passing through the insitu-generated barrier. In some embodiments, the patterning step mayfurther include forming a first module of the plurality of insitu-generated modules having a length of at least 40% of the length ofthe in situ-generated barrier, where the in situ-generated barrier has alength along the length of the channel. The in situ-generated barrierfurther has a first end and a second end. The first end of the insitu-generated barrier may be proximal to a first fluidic inlet and/or asecond fluidic inlet. In some embodiments, the step of forming an insitu-generated barrier may further include forming each of a remainderof the plurality of in situ-generated barrier modules having a length nogreater than 20% of the length of the in situ-generated barrier. In yetother embodiments, the step of forming an in situ-generated barrier mayfurther include forming the in situ-generated barrier, where the insitu-generated barrier is not porous to a flow of the first or secondfluidic medium at the first end and is porous to at least a portion ofthe flow of the first or the second fluidic medium at a point that is atleast 40% of the length of the in situ-generated barrier.

In various embodiments of the method, the method may further include thestep of reducing at least a portion of the in situ-generated barrier byapplication of increased fluidic flow in the flow region, hydrolysis,proteolysis, osmotic change, temperature change of the in situ-generatedbarrier, or optical illumination. The at least a portion of the insitu-generated barrier may be reduced by reducing a size of the barrier.In various embodiments, when the in situ-generated barrier includes theplurality of in situ-generated modules, the in situ-generated barriermay be at least reduced by removing at least a portion of the pluralityof in situ-generated barrier modules. Removing at least a portion of theplurality of the in situ-generated barrier modules may include removingone or any number of the plurality of in situ-generated barrier modules.

In various embodiments of the method, the method may further includemixing the first fluidic medium and the second fluidic medium to anextent dependent upon a location along the microfluidic channel, therebyforming a plurality of differing compositions for each of the firstfluidic medium and the second fluidic medium. In some embodiments, themethod may further include the step of growing each of the one or morecells in each of the first plurality and the second plurality ofsequestration pens clonal in the presence of a differing composition offluidic medium depending on a location of the one or more cells alongthe microfluidic channel.

In various embodiments of the method, the method may further include thestep of assaying the one or more cells in each sequestration pen of thesecond plurality of sequestration pens. In various embodiments of themethod, the method may further include the step of assaying the newclonal populations in each sequestration pen of the second plurality ofsequestration pens.

The assaying step may include flowing in a third fluidic medium into thesecond side of the channel, where the third fluidic medium includesreagents for assaying the one or more cells in each respective pen ofthe second plurality of sequestration pens. The reagents for assayingmay include one or more of chemical reagents, biological reagents,feeder cells, stimulatory cells, reporter cells, reporter molecules, andbeads. The beads may include chemical reagents, biological reagents,stimulatory reagents, or reporter molecules.

In various embodiments, the assaying step may further includeidentifying at least one cell in the second plurality of sequestrationpens having a selected characteristic. In other embodiments, theassaying step may further include identifying at least one new clonalpopulation having the selected characteristic and exporting the newclonal population so identified.

In various embodiments of the method, the method may further include thestep of exporting the at least one cell having a selected characteristics from the microfluidic device. The method may further include exportingthe clonal population from the pens of the first plurality ofsequestration pens from the microfluidic device. The method may furtherinclude the step of exporting cells that do not have the selectedcharacteristic in the second plurality of sequestration pens from themicrofluidic device. In various embodiments of the method, the methodmay further include the step of removing the in situ-generated barrierbefore exporting a cell.

Prototyping.

In another aspect, a method of rapid microfluidic device prototyping isprovided, including the steps of: providing the microfluidic device,where the device includes an enclosure including a substrate and a flowregion; introducing a first fluidic medium into the enclosure of themicrofluidic device; introducing a flowable polymer into the enclosure;selecting at least one area of the enclosure; activating solidificationof the polymer at each selected area, such that a pattern of activationresults in the formation of an in situ-generated test structure; andtesting the in situ-generated test structure for use in manipulatingmicro-objects in the microfluidic device. In some embodiments, when thetest structure does not pass the testing, then the test structure may beremoved. In some embodiments, at least a portion of the test structuremay be removable by application of increased fluidic flow in the flowregion, hydrolysis, proteolysis, osmotic change, temperature change tothe test structure, or optical illumination. In some embodiments, whenthe at least a portion of the test structure is removable by temperaturechange, then temperature change may be performed by opticallyilluminating a thermal pad on the substrate underlying the insitu-generated test structure. In various embodiments of the method, themethod may further include the step of patterning a second insitu-generated test structure having adjusted properties based on aresult of the testing step.

For all Methods:

In various embodiments of any of the methods described herein, theflowable polymer may include a synthetic polymer, a modified syntheticpolymer, or a biological polymer. The biological polymer may be light ortemperature activatable. The synthetic polymer modifications may includesize modification motifs, cleavage motifs, reactive terminal moieties,and/or cell recognition motifs, in any combination. In variousembodiments, the polymer may include at least one of a polyethyleneglycol, modified polyethylene glycol, polylactic acid (PLA), modifiedpolylactic acid, polyglycolic acid (PGA), modified polyglycolic acid,polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination. In other embodiments, the polymer may include at least oneof a polyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone(PCL), modified polycaprolactone, fibronectin, modified fibronectin,collagen, modified collagen, laminin, modified laminin, polysaccharide,modified polysaccharide, or a co-polymer in any combination. In someembodiments, the flowable polymer may include at least one of apolyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone(PCL), modified polycaprolactone, fibronectin, modified fibronectin,collagen, modified collagen, laminin, modified laminin, polysaccharide,modified polysaccharide, or a co-polymer in any combination.

For all Methods:

In various embodiments of any of the methods described herein, theflowable polymer may include a modified polyethylene glycol polymer. Inother embodiments, the modified polyethylene glycol polymer may includea star polymer. In some embodiments, the modified polyethylene glycolpolymer may include diacrylate moieties. In various embodiments, themodified polyethylene glycol polymer may include photocleavablemoieties.

For all Methods:

In various embodiments of any of the methods described herein, the stepof activating solidification of the flowable polymer may includeoptically illuminating a selected area of the substrate, therebypolymerizing the polymer. Polymerizing the flowable polymer may form asolidified polymer network. The step of activating solidification of theflowable polymer may further include introducing a photoactivatablepolymerization initiator.

For all Methods:

In various embodiments of any of the methods described herein, the stepof activating solidification of the flowable polymer may includechanging a temperature at a selected area of the substrate, and therebygelling the polymer. Gelling the flowable polymer may form a solidifiedpolymer network. The step of changing temperature at the selected areaof substrate may further include optically illuminating a thermal pad onthe substrate.

For all Methods:

In various embodiments of any of the methods described herein, at leasta portion of the in situ-generated isolation structure/isolationmodules/barrier/barrier modules may be removable by increasing flow of afluidic medium through the flow region; introducing a hydrolytic agentinto the flow region; introducing a proteolytic agent into the flowregion; introducing a fluidic medium into the flow region thatincreases/decreases an osmolality within the flow region; changing atemperature of the in situ-generated isolation structure; or opticallyilluminating the isolation structure. In some embodiments when the atleast a portion of the in situ-generated isolation structure/isolationmodules/barrier/barrier modules is removable by temperature change, thenthe temperature may be changed by optical illumination of a thermal padon the substrate underlying the barrier.

For all Methods:

In various embodiments of any of the methods described herein, thesubstrate may be configured to generate a dielectrophoretic (DEP) forceupon a micro-object in a fluidic medium within the enclosure. Thesubstrate configured to generate the DEP force may be opticallyactuated. In various embodiments of the method, the substrate may beconfigured to generate an electro-wetting force on a droplet within theenclosure. The electro-wetting forces may be optically actuated.

For all Methods:

In various embodiments, at least one inner surface of the enclosure ofthe microfluidic device may include a conditioned surface. The at leastone inner surface may include a surface of the substrate. In someembodiments, substantially all the inner surface of the enclosure mayinclude a conditioned surface. The conditioned surface may be acovalently modified surface. In some embodiments, the covalentlymodified surface may be hydrophilic. In some embodiments of the method,the method may further include a step of providing a conditioned surfaceto at least one inner surface of the enclosure. The step of providing aconditioned surface may be performed before introducing anymicro-objects, biological cells or a flowable polymer.

Kits.

In yet another aspect, a kit is provided, for isolating a micro-objectwithin a microfluidic device, comprising a microfluidic devicecomprising an enclosure comprising a substrate and a flow region locatedwithin the enclosure and a flowable polymer solution, wherein thepolymer is capable of polymerization and/or thermally induced gelling.The microfluidic device may be any microfluidic device described hereinand may have any combination of features, components and dimensions. Thekit may further include a photoactivatable polymerization initiator.

In another aspect, a kit for assaying cells of a clonal population isprovided, including a microfluidic device having an enclosure having asubstrate; a flow region comprising a channel located within theenclosure; a first plurality of sequestration pens disposed adjacent toeach other on a first side of the channel; and a second plurality ofsequestration pens disposed adjacent to each other on a second opposingside of the channel, and a flowable polymer solution, wherein thepolymer is capable of polymerization and/or thermally induced gelling.In various embodiments, the microfluidic device may further include abarrier separating the first side of the channel from the second side ofthe channel. The barrier may be an in situ-generated barrier. In variousembodiments, the barrier may not be an in situ-generated barrier and maybe punctuated by at least one gap aligned between a proximal opening tothe channel of the first pen of the first plurality of pens and aproximal opening to the channel of a first pen of the second pluralityof pens. This kit including a microfluidic device having a substrate; aflow region comprising a channel located within the enclosure; a firstplurality of sequestration pens disposed adjacent to each other on afirst side of the channel; and a second plurality of sequestration pensdisposed adjacent to each other on a second opposing side of the channelmay have any combination of features, components or dimensions for themicrofluidic device as described herein.

In various embodiments of any of the kits described herein, the polymermay include a synthetic polymer, a modified synthetic polymer, or abiological polymer. The biological polymer may be light or temperatureactivatable. The synthetic polymer modifications may include sizemodification motifs, cleavage motifs, reactive terminal moieties, and/orcell recognition motifs, in any combination. In various embodiments, thepolymer may include at least one of a polyethylene glycol, modifiedpolyethylene glycol, polylactic acid (PLA), modified polylactic acid,polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide(PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm),modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modifiedpolyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination. In other embodiments, the polymer may include at least oneof a polyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone(PCL), modified polycaprolactone, fibronectin, modified fibronectin,collagen, modified collagen, laminin, modified laminin, polysaccharide,modified polysaccharide, or a co-polymer in any combination. In someembodiments, the flowable polymer may include at least one of apolyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone(PCL), modified polycaprolactone, fibronectin, modified fibronectin,collagen, modified collagen, laminin, modified laminin, polysaccharide,modified polysaccharide, or a co-polymer in any combination.

In various embodiments of any of the kits described herein, the flowablepolymer may include a modified polyethylene glycol polymer. In otherembodiments, the modified polyethylene glycol polymer may include a starpolymer. In some embodiments, the modified polyethylene glycol polymermay include diacrylate moieties. In various embodiments, the modifiedpolyethylene glycol polymer may include photocleavable moieties.

In various embodiments of any of the kits described herein, the kit mayfurther include a reagent configured to covalently modify at least oneinternal surface of the microfluidic device, which may produce anycovalently modified internal surface as described herein. The variouscomponents of the kit may be packaged in separate containers.

EXPERIMENTAL

System and Microfluidic Device: System and Microfluidic Device:

Manufactured by Berkeley Lights, Inc. The system included at least aflow controller, temperature controller, fluidic medium conditioning andpump component, light source for light activated DEP configurations,microfluidic device, mounting stage, and a camera. The sequestrationpens have a volume of about 2×10⁷ cubic microns.

Priming Procedure:

250 microliters of 100% carbon dioxide was flowed in at a rate of 3microliters/sec. This was followed by 250 microliters of PBS, flowed inat 3 microliters per second. The final step of priming included 250microliters of culture medium containing 0.1% Pluronic® F27 (LifeTechnologies® Cat# P6866), flowed in at 3 microliters/sec.

Example 1. Installation of Isolation Structures Across Several Pens

Hydrogel preparation: A prepolymer solution was made combiningpolyethylene glycol diacrylate (PEGDA), 5 kDa (Laysan Bio, Cat. #ACRL-PEG_ACRL-5000-1 g), 10% w/v, and 1.2% w/v IGRACURE® 2959 (Ciba®,Sigma Aldrich Cat. #410896) photoinitiator in Dulbecco's phosphatebuffer saline.

Culture medium: Hybridoma-SFM (Life Technologies, Cat. #12045-076); 10%Fetal Bovine Serum; 1% Penicillin-streptomycin (100000 U/mL, LifeTechnologies Cat #15140-163); 1 mM MEM Non-Essential Amino Acid (LifeTechnologies Cat #10370-088); 2 mM GlutaMAX (Life Technologies Cat#35050-079); 1 mM Sodium Pyruvate (Life Technologies Cat #11360-070).

The microfluidic device was primed according to the general procedureabove. Culture medium was then flowed in for 5 minutes. Cells wereloaded into the microfluidic device and then introduced into thesequestration pens using gravity.

Prepolymer solution (60 microliters) was flowed into the microfluidicdevice at 2 microliter/sec and allowed to exchange with media presentwithin the sequestration pens for 5 minutes. Photoinitiatedpolymerization of selected regions (25×600 microns) within the channel,just above selected sequestration pens containing cells, was performedusing 100 microW at 340 nm+/−20 nm, introducing isolation structuresover the openings of several pens, for 7.5 sec. After completion ofphotopolymerization, culture medium was perfused at 1 microliter/sec for10 minutes to remove remaining prepolymer from the sequestration pensand flow channel. FIG. 5A shows a brightfield image of the region wherean isolation structure 520 was introduced. The solidified polymernetwork 520 is not visible under this illumination but cells 530 areclearly present in pens isolated by the isolation structure.

The entire microfluidic device was then maintained over 2 days withcontinued perfusion of media at 0.02 microliters/sec. At the end of theculturing period, the sequestration pens having an isolation structurepreventing exit of cells within the pens was visualized again, using aFITC overlay to the brightfield illumination, which is shown in FIG. 5B.Under this illumination condition, the isolation structure 520 wasclearly visible. The isolation structure prevented any cells in the pensfrom exiting. The cells 530 in the sequestration pens were still viableand demonstrated the continued ability to grow and divide.

Example 2. Introduction and Removal of Photocleavable Hydrogel Barrierswithin a Microfluidic Device

A photocleavable PEG diacrylate having a structure of Formula 1 wassynthesized.

The synthesis protocol of Kloxin et al. (Kloxin, A. M.; Tibbitt, M. W.;Anseth, K. S. Synthesis of photodegradable hydrogels as dynamicallytunable cell culture platforms. Nat. Protoc. 2010, 5 (12), 1867-1887)was used to synthesize the compound of Formula I, with modifications tomore rigorously exclude oxygen. Preparation and handling of solutions ofthe photocleavable PEG diacrylate was performed under low lightconditions for all experiments. The reaction product was used as is forall procedures.

Prepolymer solution: The prepolymer solution was prepared using 7.5% w/vPEG diacrylate M_(n) 700 (Sigma Aldrich Cat. #455008), 3.5% w/vphotodegradable PEG diacryrlate (Formula I); and 1% v/v H-NU 605 ILphotoinitiator (Spectra group Ltd) in phosphate bufferedsaline/deionized water (1:3 ratio).

Introduction of Barriers:

A microfluidic device having sequestration pens as above was primedprior to introduction of the prepolymer solution. The prepolymersolution was flowed in and allowed to diffuse into the sequestrationpens from the flow channel. Introduction of precisely and selectivelyplaced in situ-generated barriers was performed using exposure to 458 nmillumination (458 filtered visible light), with a power of approximately4.5 nW/cm². As shown in FIG. 21A, increasing exposure times producedincreasingly dense in situ-generated barriers, blocking portions of theconnection region of the selected pens and extending into the flowchannel. In situ-generated barriers 2120-2128, from left to right ofFIG. 21A, have the following exposure times:

TABLE 1 Exposure times for in situ-generated barriers. 2120 2121 21222123 2124 2125 2126 2127 10 sec 15 sec 30 sec 45 sec 60 sec 90 sec 120sec 180 secGels sufficiently formed to prevent cells from exiting the pen wereobserved for all timepoints greater than 10 sec exposure. Gels formedwith approximately 60 sec of exposure were very dense.

Removal of Barriers:

Barriers formed as above with in a microfluidic device, and storedovernight at 4 C, were exposed to illumination from an Omnicure Series2000 lamp having a 383 short pass filter installed, for two minuteexposures. As shown in FIGS. 21B and 21C, a barrier formed using a 30sec exposure (e.g., barriers2128 (Control) and 2129), was completelydegraded using the 2 minute exposure (2129″, no barrier remaining),while control barrier 2128 still remained. Increasingly dense barrierswere removable upon longer exposure (data not shown).

Example 3. Introduction of Photocleavable Hydrogel Barriers in Presenceof Cells

Cells.

OKT3 mouse myeloma (ATCC®, CRL-8001™).

Culture Medium.

IMDM (LifeTech™, 12440-061), 20% FBS (Seradigm), 10000 U/mlpenicillin-streptomycin (LifeTech™, 15140-163).

Cell Penning.

OKT3 cells were introduced into a primed microfluidic device and placedinto sequestration pens using optically actuated dielectrophoresisforces (optoelectronic tweezers (OET)), 5 volts applied field, moving at8 microns/sec. The device was kept at 37° C. until the start ofprepolymer introduction. The microfluidic device was flushed ×3 withculture medium (250 microliters at 3 microliters/sec).

Prepolymer Solution Preparation.

All photosensitive compounds prepared in low light conditions. 160microliters of prepolymer hydrogel solution was prepared to a finalconcentration of 3.75% w/v Ac star PEG star solid (4 arm PEG acrylate(10 k MW) from Laysan Bio (#4arm-PEG-ACRYL-10 k-1 g), 1.25% w/vphotodegradable PEG diacrylate (Formula I, synthesized as describedabove in Example 2); and 1% v/v H-NU 605 IL photoinitiator (Spectragroup Ltd) in culture medium containing 0.1% Pluronic® F27 (LifeTechnologies® Cat# P6866).

A solution of H-NU 605 IL photoinitiator at 1% v/v in culture mediumcontaining 0.1% Pluronic® F27 (Life Technologies® Cat# P6866) was madeby vortexing and warming at 35° C. until fully solubilized.

Introducing In Situ-Generated Barriers for Isolation of Selected Cells.

In low light conditions a primed microfluidic device was loaded with 140microliters prepolymer solution at 0.05 microliter/sec. This rateallowed diffusion of prepolymer into pens of the microfluidic device.After loading the prepolymer, a 60 sec or 120 sec exposure to UV light(Omnicure® Series 2000, Lumen Dynamics) was used to initiate polymersolidification at the top of selected pens containing cells.

After solidification was initiated, a set of rinses with culture mediumcontaining 0.1% Pluronic® F27 (Life Technologies® Cat# P6866) were usedto remove excess soluble polymers and initiator in the channel of themicrofluidic device, including 1×250 microliters at 3 microliters/sec atroom temperature; 3×250 microliters at 3 microliters/sec at 37° C. Thephotograph of FIG. 22 was taken at this timepoint.

As depicted in FIG. 22, in situ-generated isolation structures wereinstalled specifically and selectively at sequestration pen openings,without disrupting cell membranes. Some cells are highlighted in whitecircles within the isolated sequestration pens.

Recitation of Some Embodiments of the Microfluidic Devices, Methods andKits.

1. A microfluidic device including: an enclosure including: a substrate;a flow region located within the enclosure; and at least one insitu-generated isolation structure disposed on the substrate, whereinthe at least one in situ-generated structure includes a solidifiedpolymer network.

2. The microfluidic device of embodiment 1, wherein the solidifiedpolymer network includes a photoinitiated polymer.

3. The microfluidic device of embodiment 1 or 2, wherein the solidifiedpolymer network does not include a silicone polymer.

4. The microfluidic device any one of embodiments 1 to 3, wherein all orat least part of the at least one in situ-generated isolation structureconsists of the solidified polymer network.

5. The microfluidic device of any one of embodiments 1-4, wherein thesolidified polymer network includes a thermosensitive polymer.

6. The microfluidic device of embodiment 5, wherein the device furtherincludes a thermal pad disposed on the substrate at a location beneaththe at least one in situ-generated isolation structure.

7. The microfluidic device of any one of embodiments 1 to 6, wherein thedevice further includes at least one sequestration pen.

8. The microfluidic device of embodiment 7, wherein the at least onesequestration pen includes an isolation region and a connection region,the connection region having a proximal opening to the flow region and adistal opening to the isolation region.

9. The microfluidic device of embodiment 8, wherein the proximal openingof the sequestration pen to the flow region is oriented substantiallyparallel to a flow of fluidic medium in the flow region.

10. The microfluidic device of any one of embodiments 1 to 9, whereinthe flow region includes a microfluidic channel.

11. The microfluidic device of any one of embodiments 1 to 10, whereinthe device includes a plurality of sequestration pens.

12. The microfluidic device of embodiment 11, wherein the flow regionincludes a microfluidic channel, and wherein the plurality ofsequestration pens is aligned in a row, with each sequestration pen ofthe plurality opening off a common side of the microfluidic channel.

13. The device of any one of embodiments 1-12, wherein the insitu-generated isolation structure is porous to a flow of fluidicmedium.

14. The microfluidic device of any one of embodiments 1-13, wherein thein situ-generated isolation structure is configured to retain one ormore of a plurality of micro-objects.

15. The microfluidic device of any one of embodiments 1-14, wherein theat least one in situ-generated isolation structure includes a pluralityof in situ-generated isolation modules disposed in the flow region,wherein the in situ-generated isolation modules of the plurality areconfigured to substantially restrict passage of micro-objects into, outof, and/or through the at least one isolation structure in a sizedependent manner.

16. The microfluidic device of embodiment 15, wherein each insitu-generated isolation module of the plurality is spaced apart fromthe other in situ-generated isolation modules of the plurality such thatmicro-objects having a diameter of 5 microns or greater aresubstantially prevented from passing into, out of, and/or through the atleast one in situ-generated isolation structure.

17. The microfluidic device of embodiment 16, wherein the insitu-generated isolation modules of the plurality are configured toallow a first type of biological micro-object to pass into, out of,and/or through the at least one isolation structure and substantiallyprevent a second type of biological micro-object from passing into, outof, and/or through the at least one isolation structure.

18. The microfluidic device of any one of embodiments 1-17, wherein themicrofluidic device further includes a plurality of in situ-generatedisolation structures.

19. A microfluidic device including: an enclosure including: a flowregion including a microfluidic channel; a sequestration pen, whereinthe sequestration pen opens off of the microfluidic channel; and an insitu-generated isolation structure comprising an in situ-generatedbarrier disposed on the substrate, the in situ-generated barrierincluding a solidified polymer network.

20. The microfluidic device of embodiment 19, wherein the solidifiedpolymer network includes a photoinitiated polymer.

21. The microfluidic device of embodiment 19 or 20, wherein thesolidified polymer network does not include a silicone polymer.

22. The microfluidic device of any one of embodiments 19 to 21, whereinthe in situ-generated barrier at least partially blocks the microfluidicchannel and/or the sequestration pen.

23. The microfluidic device of any one of embodiments 19 to 22, whereinthe in situ-generated barrier is disposed within the connection regionof the sequestration pen.

24. The microfluidic device of embodiment 23, wherein the insitu-generated barrier has a width that extends across at least part ofa width W_(con) of the connection region, and wherein the insitu-generated barrier is configured to substantially block entry and/orexit of at least one micro-object into and/or from the sequestrationpen.

25. The microfluidic device of embodiment 24, wherein the width of thein situ-generated barrier is about 5 microns to about 20 microns.

26. The microfluidic device of any one of embodiments 23 to 25, whereina portion of the in situ-generated barrier extends from the connectionregion into the microfluidic channel.

27. The microfluidic device of embodiment 26, wherein the portion of thein situ-generated barrier extending into the channel includes less than50% of a volume of the barrier.

28. The microfluidic device of any one of embodiments 19 to 27, whereinthe in situ-generated barrier is disposed in the channel.

29. The microfluidic device of embodiment 28, wherein the insitu-generated barrier is located adjacent to one edge of the proximalopening of the sequestration pen.

30. The microfluidic device of embodiment 28 or 29 further including aplurality of sequestration pens, wherein the plurality of sequestrationpens form a row, and wherein the in situ-generated barrier is locatedadjacent to a distal edge of the proximal opening of a sequestration penlocated at the end of the row of sequestration pens.

31. The microfluidic device of any one of embodiments 28 to 30, whereinthe barrier prevents at least one subset of a plurality of micro-objectsfrom moving past the in situ-generated barrier in the channel, whereinthe plurality of micro-objects have a diameter in a range from 1 micronto 20 microns.

32. The microfluidic device of embodiment 31, wherein the insitu-generated barrier includes a plurality of in situ-generated barriermodules disposed on the substrate in the channel.

33. The microfluidic device of any one of embodiments 19 to 32, whereinthe in situ-generated barrier is porous to a flow of fluidic medium.

34. The microfluidic device of embodiment 28 or 29 further including aplurality of sequestration pens, wherein the plurality of sequestrationpens form a row, and wherein the in situ-generated barrier is disposedadjacent to an edge of the proximal opening of a selected sequestrationpen of row of sequestration pens.

35. The microfluidic device of any one of embodiments 19 to 28 furtherincluding a plurality of sequestration pens, wherein the plurality ofsequestration pens form a row, and wherein the in situ-generated barrierblocks the proximal openings of at least two contiguous sequestrationpens.

36. The microfluidic device of any one of embodiments 19 to 21, furtherincluding: a first plurality of sequestration pens disposed in a row,wherein each sequestration pen of the first plurality opens off a firstside of the microfluidic channel; and a second plurality ofsequestration pens disposed in a row, wherein each sequestration pen ofthe second plurality opens off a second opposing side of themicrofluidic channel, wherein the in situ-generated barrier is disposedalong a length of the microfluidic channel, dividing the microfluidicchannel into a first sub-channel configured to provide a first sub-flowof fluidic medium past the first plurality of sequestration pens and asecond sub-channel configured to provide a second sub-flow of fluidicmedium past the second plurality of sequestration pens, wherein the insitu-generated barrier prevents cells from moving from the firstsub-channel to the second sub-channel, and vice versa.

37. The microfluidic device of embodiment 36, wherein the insitu-generated barrier includes a plurality of in situ-generated barriermodules.

38. The microfluidic device of embodiment 37, wherein the insitu-generated barrier modules are configured to fill one or more gapsin a barrier that is not in situ-generated.

39. The microfluidic device of any one of embodiments 36 to 38, whereinthe in situ-generated barrier is porous to a flow of fluidic medium.

40. The microfluidic device of any one of embodiments 36 to 39, whereinthe first side of the microfluidic channel is configured to receive afirst fluidic medium, and the second side of the microfluidic channel isconfigured to receive a second fluidic medium.

41. The microfluidic device of any one of embodiments 36 to 40, whereinthe in situ-generated barrier prevents a micro-object having a diametergreater than 1 μm from moving from the first side of the microfluidicchannel to the second side of the microfluidic channel, or vice versa.

42. The microfluidic device of any one of embodiments 1-41, wherein thesolidified polymer network includes a synthetic polymer, a modifiedsynthetic polymer, or a biological polymer.

43. The microfluidic device of embodiment 42, wherein the syntheticpolymer modifications include size modification motifs, cleavage motifs,reactive terminal moieties, or cell recognition motifs.

44. The microfluidic device of any one of embodiments 1 to 43, whereinthe solidified polymer network includes at least one of a polyethyleneglycol, modified polyethylene glycol, polylactic acid (PLA), modifiedpolylactic acid, polyglycolic acid (PGA), modified polyglycolic acid,polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination.

46. The microfluidic device of any one of embodiments 1 to 45, whereinthe solidified polymer network includes at least one of a polyethyleneglycol, modified polyethylene glycol, polylactic acid (PLA), modifiedpolylactic acid, polyglycolic acid (PGA), modified polyglycolic acid,polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid(PAA), modified polyacrylic acid, polycaprolactone (PCL), modifiedpolycaprolactone, fibronectin, modified fibronectin, collagen, modifiedcollagen, laminin, modified laminin, polysaccharide, modifiedpolysaccharide, or a co-polymer in any combination.

47. The microfluidic device of any one of embodiments 1-46, wherein thesolidified polymer network includes a modified polyethylene glycolpolymer.

48. The microfluidic device of embodiment 47, wherein the modifiedpolyethylene glycol polymer includes a star polymer.

49. The microfluidic device of embodiment 47 or 48, wherein the modifiedpolyethylene glycol polymer includes diacrylate moieties.

50. The microfluidic device of any one of embodiments 47 to 49, whereinthe modified polyethylene glycol polymer includes photocleavablemoieties.

51. The microfluidic device of any one of embodiments 42 to 50, whereinthe solidified polymer network is susceptible to degradation byhydrolysis, proteolysis, osmotic change, temperature change or opticalillumination.

52. The microfluidic device of any one of embodiments 42 to 51, whereinthe solidified polymer network is susceptible to displacement byincreased fluidic flow.

53. The microfluidic device of any one of embodiments 1 to 52, whereinat least one inner surface of the enclosure includes a conditionedsurface.

54. The microfluidic device of embodiment 53, wherein the at least oneinner surface includes a surface of the substrate.

55. The microfluidic device of embodiment 53 or 54, wherein theconditioned surface is a covalently modified surface.

56. The microfluidic device of embodiment 55, wherein the covalentlymodified surface is hydrophilic.

57. The microfluidic device of any one of embodiments 1-56, wherein thesubstrate is configured to generate dielectrophoresis (DEP) forceswithin the enclosure.

58. The microfluidic device of embodiment 57, wherein the DEP forces areoptically actuated.

59. The microfluidic device of any one of embodiments 1 to 59, wherein acover of the microfluidic device is transparent.

60. A method of isolating a micro-object in a microfluidic device,including the steps of: introducing a first fluidic medium including aplurality of micro-objects into an enclosure of the microfluidic device,the enclosure including a substrate and a flow region; introducing asolution including a flowable polymer into the enclosure; activatingsolidification of the flowable polymer at at least one selected area ofthe flow region, thereby forming an in situ-generated isolationstructure; and isolating at least one of the plurality of micro-objectswith the in situ-generated isolation structure.

61. The method of embodiment 60, wherein the step of introducing asolution including a flowable polymer is performed before the step ofintroducing the first fluidic medium including the plurality ofmicro-objects

62. The method of embodiment 61 wherein the step of initiatingsolidification of the flowable polymer includes optically illuminatingthe at least one selected area of the flow region, and further whereinthe step of solidification of the flowable polymer includes polymerizingpolymers of the flowable polymer to form a polymer network.

63. The method of any one of embodiments 60 to 62, wherein the step ofintroducing a flowable polymer further includes introducing aphotoactivatable polymerization initiator.

64. The method of any one of embodiments 60 to 62, wherein the step ofinitiating solidification of the flowable polymer includes changing atemperature at the at least one selected area of the substrate, andfurther wherein the step of solidification of the polymer includesgelling the polymer to form a polymer network.

65. The method of any one of embodiments 60 to 64, further includingprocessing a remainder of the plurality of micro-objects.

66. The method of any one of embodiments 60 to 65, further includingexporting a remainder of the plurality of micro-objects from themicrofluidic device.

67. The method of any one of embodiments 60 to 66, wherein the at leastone micro-object isolated by the in situ-generated isolation structureis a selected portion of the plurality of micro-objects.

68. The method of any one of embodiments 60 to 67, further including thestep of: reducing or removing the in situ-generated isolation structureby: increasing flow of a fluidic medium through the flow region;introducing a hydrolytic agent into the flow region; introducing aproteolytic agent into the flow region; introducing a fluidic mediuminto the flow region that increases/decreases an osmolality within theflow region; changing a temperature of the in situ-generated isolationstructure; or optically illuminating the isolation structure, andthereby releasing the at least one micro-object from the insitu-generated isolation structure.

69. The method of embodiment 68, wherein the step of changing thetemperature further includes optically illuminating a thermal pad on thesubstrate adjacent to or under the in situ-generated isolationstructure.

70. The method of embodiment 68 or 69, further including the step ofexporting the at least one released micro-object from the microfluidicdevice.

71. The method of any one of embodiments 60 to 70, wherein the insitu-generated isolation structure is at least partially porous to aflow of a fluidic medium.

72. The method of any one of embodiments 60 to 71, wherein the step ofactivating solidification of the flowable polymer includes forming an insitu-generated isolation structure comprising an in situ-generatedbarrier configured to prevent passage of the at least one micro-objectinto, out of, or through the in situ-generated isolation structure.

73. The method of any one of embodiments 60 to 72, wherein the step ofactivating solidification of the flowable polymer includes forming aplurality of in situ-generated barriers configured to prevent passage ofsub-sets of the plurality of micro-objects into, out of, or through thein situ-generated isolation structure.

74. The method of embodiment 73, further including the step of reducingor removing one or more of the plurality of in situ-generated barriersby: increasing flow of a fluidic medium through the flow region;introducing a hydrolytic agent into the flow region; introducing aproteolytic agent into the flow region; introducing a fluidic mediuminto the flow region that increases/decreases an osmolality within theflow region; changing a temperature of the in situ-generated barriers;or optically illuminating the in situ-generated barriers, and therebyreleasing one or more sub-sets of the at least one micro-object from thein situ-generated isolation structure.

75. The method of embodiment 74, wherein the step of reducing orremoving one or more of the in situ-generated barriers includesoptically illuminating the in situ-generated barrier.

76. The method of embodiment 74, wherein the step of changing thetemperature of the one or more in situ-generated barriers furtherincludes optically illuminating a thermal pad on the substrate adjacentor under the one or more in situ-generated barriers.

77. The method of any one of embodiment 74 to 76, further including thestep of exporting the one or more sub-sets of the at least one releasedmicro-object from the microfluidic device.

78. The method of any one of embodiments 72 to 77, wherein the step ofactivating solidification further includes forming an in situ-generatedisolation structure comprising an in situ-generated barrier including aplurality of in situ-generated barrier modules, each of the plurality ofin situ-generated barrier modules spaced apart from each other at adistance preventing the at least one micro-object from passing throughthe in situ-generated isolation structure.

79. The method of embodiment 78, further including the step of spacingthe plurality of in situ-generated barrier modules apart from each otherthereby preventing at least one subset of micro-objects of the pluralityof micro-objects from passing through the in situ-generated isolationstructure.

80. The method of embodiment 79, wherein the at least one subset ofmicro-objects includes at least one type of biological cell.

81. The method of any one of embodiments 60-80, wherein the enclosure ofthe microfluidic device further includes at least one sequestration penincluding an isolation region and a connection region, the connectionregion having a proximal opening to the flow region and a distal openingto the isolation region.

82. The method of embodiment 81, wherein the enclosure includes aplurality of sequestration pens.

83. The method of embodiment 82, wherein the plurality of sequestrationpens is disposed in a row, and the proximal opening of each of theplurality of sequestration pens are disposed contiguously to each other.

84. The method of embodiment 82 or 83, wherein the flow region includesa microfluidic channel and the proximal opening of each of the pluralityof sequestration pens opens off of one side of the microfluidic channel.

85. The method of any one of embodiments 81 to 84, wherein the step ofactivating solidification is performed inside a sequestration pen.

86. The method of embodiment 85, wherein the step of activation ofsolidification is performed within the isolation region or theconnection region.

87. The method of embodiment 85 or 86, wherein the step of activatingsolidification of the flowable polymer generates an in situ-generatedisolation structure including an in situ-generated barrier in theconnection region.

88. The method of any one of embodiments 81 to 87, further including thestep of processing a remainder of the plurality of micro-objects notisolated by the in situ-generated barrier.

89. The method of any one of embodiments 81 to 87, further including thestep of exporting a remainder of the plurality of micro-objects notisolated by the in situ-generated barrier from the microfluidic device.

90. The method of embodiment 89, further including the step of reducingor removing the in situ-generated barrier by increasing flow of afluidic medium through the flow region, introducing a hydrolytic agentinto the flow region, introducing a proteolytic agent into the flowregion, introducing a fluidic medium into the flow region that increasesor decreases an osmolality of the fluidic medium, changing a temperatureof the in situ-generated barrier, or optically illuminating the barrier,thereby releasing the at least one micro-object from isolation.

91. The method of embodiment 90, wherein the step of reducing orremoving the in situ-generated barrier includes optically illuminatingthe in situ-generated barrier.

92. The method of embodiment 90, wherein changing temperature furtherincludes optically illuminating a thermal pad on the substrate adjacentor under the in situ-generated barrier.

93. The method of any one of embodiments 90 to 92, further including astep of exporting the at least one micro-object from the microfluidicdevice.

94. The method of embodiment 84, wherein the step of activatingsolidification of the flowable polymer forms an in situ-generatedisolation structure including an in situ-generated barrier in thechannel.

95. The method of embodiment 94, wherein the step of activatingsolidification of the flowable polymer disposes the in situ-generatedbarrier at a proximal opening of at least one sequestration pen of theplurality of sequestration pens.

96. The method of embodiment 94 or 95, wherein the step of activatingsolidification of the flowable polymer forms an in situ-generatedbarrier sized to block the proximal openings of at least two contiguoussequestration pens.

97. The method of embodiment 96, further including a step of exporting aremainder of the plurality of micro-objects not isolated by the insitu-generated barrier from the microfluidic device.

98. The method of embodiment 94, wherein the microfluidic device furtherincludes a second plurality of sequestration pens disposed adjacent toeach other such that each sequestration pen opens off a second side ofthe microfluidic channel; and the step of activating solidification ofthe flowable polymer further includes activating solidification of theflowable polymer along a length of the microfluidic channel, therebyforming an in situ-generated isolation structure comprising an insitu-generated barrier dividing the microfluidic channel into a firstsub-channel configured to provide a first sub-flow of a fluidic mediumpast the first plurality of sequestration pens and a second sub-channelconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of pens, wherein the in situ-generated barrier preventsmicro-objects from moving from the first sub-channel to the secondsub-channel, and vice versa.

99. The method of embodiment 98, wherein the microfluidic device furtherincludes a barrier dividing the microfluidic channel into a firstsub-channel configured to provide a first sub-flow of a fluidic mediumpast the first plurality of sequestration pens and a second sub-channelconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of pens, the barrier punctuated by at least one gapaligned with a proximal opening to the first sub-channel of a firstsequestration pen of the first plurality of sequestration pens andaligned with a proximal opening to a proximal opening to the secondsub-channel of a first pen of the second plurality of sequestrationpens; and further wherein the step of activating polymerization includesactivating polymerization at the at least one gap to form at least onein situ-generated barrier in the at least one gap, thereby preventingmicro-objects from moving from the first sub-channel to the secondsub-channel, and vice versa.

100. The method of embodiment 98 or 99, wherein the step of introducingthe plurality of micro-objects further includes introducing a clonalpopulation of cells in each of the sequestration pens of the firstplurality of sequestration pens; and, for each clonal population ofcells in the first plurality of sequestration pens, moving at least onecell to a respective sequestration pen of the second plurality of pens.

101. The method of embodiment 100, wherein when the microfluidic deviceincludes the barrier punctuated by at least one gap, the step of movingthe at least one cell of each clonal population in the first pluralityof sequestration pens includes moving the at least one cell to therespective sequestration pen of the second plurality of pens through theat least one gap.

102. The method of embodiment 100 or 101, wherein the method furtherincludes a step of processing the cell(s) in the second plurality ofsequestration pens.

103. The method of any one of embodiments 60 to 102, wherein theflowable polymer includes a synthetic polymer, a modified syntheticpolymer, or a biological polymer.

104. The method of embodiment 103, wherein the synthetic polymermodifications include size modification motifs, cleavage motifs,reactive terminal moieties, or cell recognition motifs.

105. The method of any one of embodiments 60 to 104, wherein theflowable polymer includes at least one of a polyethylene glycol,modified polyethylene glycol, polylactic acid (PLA), modified polylacticacid, polyglycolic acid (PGA), modified polyglycolic acid,polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination.

106. The method of any one of embodiments 60 to 105, wherein theflowable polymer includes at least one of a polyethylene glycol,modified polyethylene glycol, polylactic acid (PLA), modified polylacticacid, polyglycolic acid (PGA), modified polyglycolic acid, polyvinylalcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA),modified polyacrylic acid, polycaprolactone (PCL), modifiedpolycaprolactone, fibronectin, modified fibronectin, collagen, modifiedcollagen, laminin, modified laminin, polysaccharide, modifiedpolysaccharide, or a co-polymer in any combination.

107. The method of any one of embodiments 60 to 106, wherein thesolidified polymer network includes a modified polyethylene glycolpolymer.

108. The method of embodiment 107, wherein the modified polyethyleneglycol polymer includes a star polymer.

109. The method of embodiment 107 or 108, wherein the modifiedpolyethylene glycol polymer includes diacrylate moieties.

110. The method of any one of embodiments 107 to 109, wherein themodified polyethylene glycol polymer includes photocleavable moieties.

111. The method of any one of embodiments 60 to 110, wherein at leastone inner surface of the enclosure includes a conditioned surface.

112. The method of embodiment 111, wherein the at least one innersurface includes a surface of the substrate.

113. The method of any one of embodiments 110 to 112, wherein theconditioned surface is a covalently modified surface.

114. The method of embodiment 113, wherein the covalently modifiedsurface is hydrophilic.

115. The method of any one of embodiments 60 to 114, wherein the step ofintroducing the plurality of micro-objects further includes usingdielectrophoresis (DEP) forces.

116. The method of any one of embodiments 66 to 115, wherein the step ofexporting one or more micro-objects of the plurality of micro-objectsfurther includes using dielectrophoresis (DEP) forces.

117. The method of embodiment 115 or 116, further including opticallyactuating the DEP forces.

118. A method of assaying a cell of a clonal population in amicrofluidic device, the method including the steps of: introducing afirst fluidic medium including a plurality of cells into an enclosure ofthe microfluidic device, the enclosure including: a substrate; a flowregion including a microfluidic channel configured to contain a fluidicmedium; a first plurality of sequestration pens disposed adjacent toeach other such that each sequestration pen of the first plurality opensoff a first side of the microfluidic channel; and a second plurality ofsequestration pens disposed adjacent to each other such that eachsequestration pen of the second plurality opens off a second opposingside of the microfluidic channel; flowing the first fluidic medium andthe plurality of cells into the channel of the microfluidic device;introducing a clonal population of cells in each of the sequestrationpens of the first plurality of sequestration pens; for each clonalpopulation of cells in the first plurality of sequestration pens, movingat least one cell to a respective sequestration pen of the secondplurality of sequestration pens; introducing a flowable polymer into thechannel; activating solidification of the flowable polymer along alength of the microfluidic channel, thereby forming an in situ-generatedbarrier dividing the microfluidic channel into a first sub-channelconfigured to provide a first sub-flow of fluidic medium past the firstplurality of sequestration pens and a second sub-channel configured toprovide a second sub-flow of fluidic medium past the second plurality ofsequestration pens, wherein the in situ-generated barrier prevents cellsfrom moving from the first sub-channel to the second sub-channel, andvice versa; flowing a second fluidic medium into the second sub-channel,wherein the second fluidic medium includes reagents for assaying thecells in the second plurality of sequestration pens; and, assaying thecell(s) in each sequestration pen of the second plurality.

119. The method of embodiment 118, wherein the in situ-generated barrierhas a length from a first end of the channel to a second end of themicrofluidic channel.

120. The method of embodiment 118 or 119, wherein each sequestration penof the first plurality of sequestration pens and of the second pluralityof sequestration pens has a proximal opening to its respective side ofthe microfluidic channel.

121. The method of any one of embodiments 118 to 120, wherein the stepof introducing the clonal population includes introducing a single cellto each of the first plurality of sequestration pens, and furtherincludes expanding the single cell to a clonal population of cells.

122. The method of embodiment 121 further including flowing fluidicmedium in the first sub-channel and flowing fluidic medium in the secondsub-channel to respective first and second outputs of the microfluidicdevice.

123. The method of any one of embodiments 118 to 122, wherein the stepof assaying includes preparing the cells in the second plurality ofsequestration pens for genotyping.

124. The method of any one of embodiments 118 to 123, wherein the stepof assaying includes determining a level of production of a biologicalproduct by the cell(s) in each sequestration pen of the second pluralityand/or by the clonal population in each sequestration pen of the firstplurality.

125. The method of any one of embodiments 118 to 124, wherein the insitu-generated barrier prevents the first sub-flow of fluidic medium inthe first sub-channel from mixing with the second sub-flow of fluidicmedium in the second sub-channel.

126. The method of any one of embodiments 118 to 125, wherein thereagents for assaying include one or more of the group includingchemical reagents, biological reagents, feeder cells, stimulatory cells,reporter cells, reporter molecules, and beads.

127. The method of embodiment 126 wherein the beads include chemicalreagents, biological reagents, stimulatory reagents, or reportermolecules.

128. The method of any one of embodiments 118 to 127, wherein theassaying step further includes identifying at least one cell of thecells in the second plurality of sequestration pens, the at least onecell including a selected characteristic.

129. The method of any one of embodiments 118 to 128, further includinga step of exporting at least one cell of the cells in the secondplurality of sequestration pens, wherein the at least one cell includesa selected characteristic.

130. The method of embodiment 128 or 129, further including a step ofexporting the respective clonal population from the respective pen ofthe first plurality of sequestration pens.

131. The method of any one of embodiments 118 to 130, further includinga step of exporting the cells in the second plurality of sequestrationpens and/or clonal populations of cells in the first plurality ofsequestration pens that do not include a selected characteristic.

132. The method of any one of claims 129 to 131, further including thestep of removing the in situ-generated barrier before exporting a cell.

133. The method of any one of embodiments 118 to 132, wherein the stepof activating solidification the polymer includes optically illuminatinga selected area of the substrate, thereby polymerizing the polymer.

134. The method of embodiment 133, wherein the step of activatingsolidification of the flowable polymer further includes introducing aphotoactivatable polymerization initiator.

135. The method of any one of embodiments 118 to 134, wherein the stepof activating solidification of the flowable polymer along a length ofthe microfluidic channel includes activating solidification of theflowable polymer at gaps of a barrier extending from a first end of themicrofluidic channel to a second end of the microfluidic channel, thebarrier separating the microfluidic channel into the first sub-channeland the second sub-channel, wherein the gaps are aligned with theproximal opening of each pen of the first plurality of sequestrationpens and the proximal opening of the respective pen of the secondplurality of sequestration pens, thereby forming an in situ-generatedbarrier preventing cells from moving from the first sub-channel to thesecond sub-channel, and vice versa.

136. The method of any one of embodiments 118 to 130, wherein the stepof activating solidification of the polymer includes changing atemperature at a selected area of the substrate, and thereby gelling thepolymer.

137. The method of any one of embodiments 118 to 136, wherein theflowable polymer includes a synthetic polymer, a modified syntheticpolymer, or a biological polymer.

138. The method of embodiment 137, wherein the synthetic polymermodifications include size modification motifs, cleavage motifs,reactive terminal moieties, and/or cell recognition motifs.

139. The method of any one of embodiments 118 to 138, wherein theflowable polymer includes at least one of a polyethylene glycol,modified polyethylene glycol, polylactic acid (PLA), modified polylacticacid, polyglycolic acid (PGA), modified polyglycolic acid,polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination.

140. The method of any one of embodiments 118 to 139, wherein theflowable polymer includes at least one of a polyethylene glycol,modified polyethylene glycol, polylactic acid (PLA), modified polylacticacid, polyglycolic acid (PGA), modified polyglycolic acid, polyvinylalcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA),modified polyacrylic acid, polycaprolactone (PCL), modifiedpolycaprolactone, fibronectin, modified fibronectin, collagen, modifiedcollagen, laminin, modified laminin, polysaccharide, modifiedpolysaccharide, or a co-polymer in any combination.

141. The method of any one of embodiments 118 to 140, wherein thesolidified polymer network includes a modified polyethylene glycolpolymer.

142. The method of embodiment 141, wherein the modified polyethyleneglycol polymer includes a star polymer.

143. The method of embodiment 141 or 142, wherein the modifiedpolyethylene glycol polymer includes diacrylate moieties.

144. The method of any one of embodiments 141 to 143, wherein themodified polyethylene glycol polymer includes photocleavable moieties.

145. The method of any one of embodiments 116 to 144, wherein the stepof introducing the clonal population of micro-objects or moving thesingle cell is performed using dielectrophoretic (DEP) forces.

146. The method of embodiment 145, further including optically actuatingthe DEP forces.

147. The method of any one of embodiments 118-146, wherein at least oneinner surface of the enclosure includes a conditioned surface.

148. The method of embodiment 147, wherein the at least one innersurface includes a surface of the substrate.

149. The method of embodiment 147 or 148, wherein the conditionedsurface is a covalently modified surface.

150. The method of embodiment 149, wherein the covalently modifiedsurface is hydrophilic.

151. A kit including a microfluidic device of any one of embodiments 1to 59, and a flowable polymer solution, wherein the polymer is capableof polymerization and/or thermally induced gelling.

152. The kit of embodiment 151, further including a photoactivatablepolymerization initiator.

153. The kit of embodiment 151 or 152, wherein the flowable polymerincludes a synthetic polymer, a modified synthetic polymer, or abiological polymer.

154. The kit of embodiment 153, wherein the modified synthetic polymerincludes size modification motifs, cleavage motifs, reactive terminalmoieties, or cell recognition motifs.

155. The kit of any one of embodiments 151 to 154, wherein the flowablepolymer includes at least one of a polyethylene glycol, modifiedpolyethylene glycol, polylactic acid (PLA), modified polylactic acid,polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide(PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm),modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modifiedpolyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination.

156. The kit of any one of embodiments 151 to 155, wherein the flowablepolymer includes at least one of a polyethylene glycol, modifiedpolyethylene glycol, polylactic acid (PLA), modified polylactic acid,polyglycolic acid (PGA), modified polyglycolic acid, polyvinyl alcohol(PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modifiedpolyacrylic acid, polycaprolactone (PCL), modified polycaprolactone,fibronectin, modified fibronectin, collagen, modified collagen, laminin,modified laminin, polysaccharide, modified polysaccharide, or aco-polymer in any combination.

157. The kit of any one of embodiments 151 to 156, wherein the flowablepolymer includes a modified polyethylene glycol polymer.

158. The kit of embodiment 157, wherein the modified polyethylene glycolpolymer includes a star polymer.

159. The kit of embodiment 157 or 158, wherein the modified polyethyleneglycol polymer includes diacrylate moieties.

160. The kit of any one of embodiments 153 to 154, wherein the modifiedpolyethylene glycol polymer includes photocleavable moieties.

161. The kit of any one of embodiments 151 to 161, further including areagent configured to covalently modify at least one internal surface ofthe microfluidic device.

162. A microfluidic device including: an enclosure including: asubstrate; a flow region including a microfluidic channel configured tocontain a fluidic medium; a first plurality of sequestration pensdisposed adjacent to each other such that each sequestration pen of thefirst plurality opens off a first side of the microfluidic channel; anda second plurality of sequestration pens disposed adjacent to each othersuch that each sequestration pen of the second plurality opens off asecond opposing side of the microfluidic channel.

163. The microfluidic device of embodiment 162, wherein the first sideof the microfluidic channel is configured to receive a first fluidicmedium, and the second side of the microfluidic channel is configured toreceive a second fluidic medium.

164. The microfluidic device of embodiment 163, wherein the firstfluidic medium is introduced into the first side of microfluidic thechannel via a first fluidic inlet and the second fluidic medium isintroduced into the second side of the microfluidic channel via a secondfluidic inlet.

165. The microfluidic device of embodiment 163 or 164, wherein the firstfluidic medium flows out of the first side of the microfluidic channelvia a first outlet and the second fluidic medium flows out of the secondside of the microfluidic channel via a second outlet.

166. The microfluidic device of any one of embodiments 162-165, whereineach sequestration pen of the first plurality and the second pluralityof sequestration pens includes an isolation region and a connectionregion, the connection region having a proximal opening to the channeland a distal opening to the isolation region.

167. The microfluidic device of embodiment 166, wherein the proximalopening of the sequestration pen to the channel is orientedsubstantially parallel to a flow of fluidic medium in the channel.

168. The microfluidic device of any one of embodiments 162-167, whereinat least one inner surface of the enclosure includes a conditionedsurface.

169. The microfluidic device of embodiment 168, wherein the at least oneinner surface includes a surface of the substrate.

170. The microfluidic device of embodiment 168 or 169, wherein theconditioned surface is a covalently modified surface.

171. The microfluidic device of any one of embodiments 168-169, whereinthe covalently modified surface is hydrophilic.

172. The microfluidic device of any one of embodiments 162-171, whereinthe substrate is configured to generate dielectrophoresis (DEP) forceswithin the enclosure.

173. The microfluidic device of embodiment 172, wherein the DEP forcesare optically actuated.

174. The microfluidic device of any one of embodiments 162-173, whereina cover of the microfluidic device is substantially transparent.

175. The microfluidic device of any one of embodiments 162-174, furtherincluding a barrier dividing the microfluidic channel into a firstsub-channel configured to provide a first sub-flow of fluidic mediumpast the first plurality of sequestration pens and a second sub-channelconfigured to provide a second sub-flow of fluidic medium past thesecond plurality of sequestration pens, wherein the barrier ispunctuated by at least one gap aligned between a proximal opening to thefirst sub-channel of the first pen of the first plurality of pens and aproximal opening to the second sub-channel of a first pen of the secondplurality of pens.

176. The microfluidic device of embodiment 175, wherein the barrier hasa length that extends from a first end of the channel to a second end ofthe channel.

177. The microfluidic device of embodiment 175 or 176, wherein thebarrier of the microfluidic device further includes a gap alignedbetween a proximal opening to the first sub-channel of each pen of thefirst plurality of pens and a proximal opening to the second sub-channelof each respective pen of the second plurality of pens, wherein thebarrier thereby includes a plurality of gaps along a length of thebarrier in the microfluidic channel.

178. The microfluidic device of embodiment 175 or 176, further includingan in situ-generated barrier closing the gap in the barrier separatingthe first sub-channel from the second sub-channel.

179. The microfluidic device of embodiment 178, further including an insitu-generated barrier closing each of the plurality of gaps of thebarrier separating the first sub-channel from the second sub-channel,wherein the barrier thereby includes a plurality of in situ-generatedbarriers.

180. The microfluidic device of any one of embodiments 162-174, furtherincluding an in situ-generated barrier, wherein the in situ-generatedbarrier is disposed along a length of the microfluidic channel, dividingthe microfluidic channel into a first sub-channel configured to providea first sub-flow of fluidic medium past the first plurality ofsequestration pens and a second sub-channel configured to provide asecond sub-flow of fluidic medium past the second plurality ofsequestration pens, wherein the in situ-generated barrier prevents cellsfrom moving from the first sub-channel to the second sub-channel, andvice versa.

181. The microfluidic device of any one of embodiments 178-180, whereinthe in situ-generated barrier includes a solidified polymer network.

182. The microfluidic device of embodiments 181, wherein the solidifiedpolymer network includes a synthetic polymer, a modified syntheticpolymer, or a biological polymer.

183. The microfluidic device of embodiment 182, wherein the syntheticpolymer modifications include size modification motifs, cleavage motifs,reactive terminal moieties, or cell recognition motifs.

184. The microfluidic device of any one of embodiments 181-183, whereinthe solidified polymer network includes at least one of a polyethyleneglycol, modified polyethylene glycol, polylactic acid (PLA), modifiedpolylactic acid, polyglycolic acid (PGA), modified polyglycolic acid,polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination.

185. The microfluidic device of any one of embodiments 181-184, whereinthe solidified polymer network includes at least one of a polyethyleneglycol, modified polyethylene glycol, polylactic acid (PLA), modifiedpolylactic acid, polyglycolic acid (PGA), modified polyglycolic acid,polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid(PAA), modified polyacrylic acid, polycaprolactone (PCL), modifiedpolycaprolactone, fibronectin, modified fibronectin, collagen, modifiedcollagen, laminin, modified laminin, polysaccharide, modifiedpolysaccharide, or a co-polymer in any combination.

186. The microfluidic device of any one of embodiments 176-180, whereinthe solidified polymer network includes a modified polyethylene glycolpolymer.

187. The microfluidic device of embodiment 186, wherein the modifiedpolyethylene glycol polymer includes a star polymer.

188. The microfluidic device of embodiment 186 or 187, wherein themodified polyethylene glycol polymer includes diacrylate moieties.

189. The microfluidic device of any one of embodiments 186 to 188wherein the modified polyethylene glycol polymer includes photocleavablemoieties.

190. A kit for assaying cells of a clonal population, including amicrofluidic device of any one of embodiments 162-177, and a flowablepolymer solution, wherein the polymer is capable of polymerizationand/or thermally induced gelling.

191. The kit of embodiment 190, further including a photoactivatablepolymerization initiator.

192. The kit of embodiment 190 or 191, wherein the flowable polymerincludes a synthetic polymer, a modified synthetic polymer, or abiological polymer.

193. The kit of embodiment 192, wherein the modified synthetic polymerincludes size modification motifs, cleavage motifs, reactive terminalmoieties, or cell recognition motifs.

194. The kit of any one of embodiments 190-193, wherein the flowablepolymer includes at least one of a polyethylene glycol, modifiedpolyethylene glycol, polylactic acid (PLA), modified polylactic acid,polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide(PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm),modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modifiedpolyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination.

195. The kit of any one of embodiments 190-194, wherein the flowablepolymer includes at least one of a polyethylene glycol, modifiedpolyethylene glycol, polylactic acid (PLA), modified polylactic acid,polyglycolic acid (PGA), modified polyglycolic acid, polyvinyl alcohol(PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modifiedpolyacrylic acid, polycaprolactone (PCL), modified polycaprolactone,fibronectin, modified fibronectin, collagen, modified collagen, laminin,modified laminin, polysaccharide, modified polysaccharide, or aco-polymer in any combination.

196. The kit of any one of embodiments 190-195, wherein the flowablepolymer includes a modified polyethylene glycol polymer.

197. The kit of embodiment 196, wherein the modified polyethylene glycolpolymer includes a star polymer.

193. The kit of embodiment 191 or 192, wherein the modified polyethyleneglycol polymer includes diacrylate moieties.

194. The kit of any one of embodiments 191 to 193, wherein the modifiedpolyethylene glycol polymer includes photocleavable moieties.

195. The kit of any one of embodiments 185 to 194, further including areagent configured to covalently modify at least one internal surface ofthe microfluidic device.

1. A microfluidic device comprising: an enclosure comprising: asubstrate; a flow region located within the enclosure; and at least onein situ-generated isolation structure disposed on the substrate, whereinthe at least one in situ-generated structure comprises a solidifiedpolymer network.
 2. The microfluidic device of claim 1, wherein thesolidified polymer network comprises a photoinitiated polymer. 3.(canceled)
 4. The microfluidic device of claim 1, wherein themicrofluidic device further comprises at least one sequestration pen,wherein the at least one sequestration pen comprises an isolation regionand a connection region, the connection region having a proximal openingto the flow region and a distal opening to the isolation region. 5.(canceled)
 6. The microfluidic device of claim 1, wherein the flowregion comprises a microfluidic channel.
 7. The microfluidic device ofclaim 1, wherein the microfluidic device comprises a plurality ofsequestration pens.
 8. The microfluidic device of claim 1, wherein thein situ-generated isolation structure is configured to retain one ormore of a plurality of micro-objects.
 9. The microfluidic device ofclaim 1, wherein the at least one in situ-generated isolation structurecomprises a plurality of in situ-generated isolation modules disposed inthe flow region, wherein the in situ-generated isolation modules of theplurality are configured to substantially restrict passage ofmicro-objects into, out of, and/or through the at least one isolationstructure in a size dependent manner. 10.-11. (canceled)
 12. Amicrofluidic device comprising: an enclosure comprising: a flow regioncomprising a microfluidic channel; a sequestration pen, wherein the atleast one sequestration pen comprises an isolation region and aconnection region, the connection region having a proximal opening tothe microfluidic channel and a distal opening to the isolation region;and an in situ-generated isolation structure comprising an insitu-generated barrier disposed on the substrate, the in situ-generatedbarrier comprising a solidified polymer network. 13.-26. (canceled) 27.The microfluidic device of claim 1, wherein the solidified polymernetwork comprises a synthetic polymer, a modified synthetic polymer, ora biological polymer. 28.-29. (canceled)
 30. The microfluidic device ofclaim 1, wherein the solidified polymer network comprises a modifiedpolyethylene glycol polymer.
 31. The microfluidic device of claim 1,wherein the solidified polymer network is susceptible to degradation byhydrolysis, proteolysis, osmotic change, temperature change, opticalillumination, or displacement by increased fluidic flow.
 32. (canceled)33. The microfluidic device of claim 1, wherein at least one innersurface of the enclosure comprises a conditioned surface.
 34. Themicrofluidic device of claim 1, wherein the substrate is configured togenerate dielectrophoresis (DEP) forces within the enclosure. 35.(canceled)
 36. A method of isolating a micro-object in a microfluidicdevice, comprising the steps of: introducing a first fluidic mediumcomprising a plurality of micro-objects into an enclosure of themicrofluidic device, the enclosure comprising a substrate and a flowregion; introducing a solution comprising a flowable polymer into theenclosure; activating solidification of the flowable polymer at at leastone selected area of the flow region, thereby forming an insitu-generated isolation structure; and isolating at least one of theplurality of micro-objects with the in situ-generated isolationstructure.
 37. The method of claim 36, wherein the step of initiatingsolidification of the flowable polymer comprises optically illuminatingthe at least one selected area of the flow region, and further whereinthe step of solidification of the flowable polymer comprisespolymerizing polymers of the flowable polymer to form a polymer network.38. The method of claim 36, wherein the step of introducing a flowablepolymer further comprises introducing a photoactivatable polymerizationinitiator. 39.-40. (canceled)
 41. The method of claim 36, furthercomprising the step of: reducing or removing the isolation structure by:increasing flow of a fluidic medium through the flow region; introducinga hydrolytic agent into the flow region; introducing a proteolytic agentinto the flow region; introducing a fluidic medium into the flow regionthat increases/decreases an osmolality within the flow region; changinga temperature of the in situ-generated isolation structure; or opticallyilluminating the isolation structure, and thereby releasing the at leastone micro-object from the in situ-generated isolation structure. 42.(canceled)
 43. The method of claim 36, wherein the in situ-generatedisolation structure is at least partially porous to a flow of a fluidicmedium.
 44. The method of claim 36, wherein the step of activatingsolidification of the flowable polymer comprises forming an insitu-generated isolation structure comprising an in situ-generatedbarrier configured to prevent passage of the at least one micro-objectinto, out of, or through the in situ-generated isolation structure.45.-47. (canceled)
 48. The method of claim 36, wherein the enclosure ofthe microfluidic device further comprises at least one sequestration pencomprising an isolation region and a connection region, the connectionregion having a proximal opening to the flow region and a distal openingto the isolation region. 49.-50. (canceled)
 51. The method of claim 48,wherein the step of activating solidification is performed inside asequestration pen.
 52. The method of claim 48, wherein the step ofactivating solidification of the flowable polymer forms an insitu-generated isolation structure comprising an in situ-generatedbarrier in the connection region of the at least one sequestration pen,in the flow region, or a combination thereof. 53.-59. (canceled)
 60. Themethod of any one of claims 36-59, wherein the flowable polymercomprises a synthetic polymer, a modified synthetic polymer, or abiological polymer.
 61. (canceled)
 62. The method of claim 36, whereinthe step of introducing the plurality of micro-objects further comprisesusing dielectrophoresis (DEP) forces. 63.-81. (canceled)
 82. A kitcomprising a microfluidic device of claim 1, and a flowable polymersolution, wherein the polymer is capable of polymerization and/orthermally induced gelling. 83.-86. (canceled)
 87. The kit of claim 82,further comprising a reagent configured to covalently modify at leastone internal surface of the microfluidic device.
 88. The microfluidicdevice of claim 12, further comprising a substrate configured togenerate dielectrophoresis (DEP) forces within the enclosure.